DESIGN, PRODUCTION, AND FUNCTIONAL AND STRUCTURAL
CHARACTERIZATION OF HIV-1 ENVELOPE PROTEIN GP41 ECTODOMAIN
By
Koyeli Banerjee

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Chemistry – Doctor of Philosophy
Quantitative Biology – Dual Major
2014

ABSTRACT
DESIGN, PRODUCTION, AND FUNCTIONAL AND STRUCTURAL CHARACTERIZATION
OF HIV-1 ENVELOPE PROTEIN GP41 ECTODOMAIN
By
Koyeli Banerjee
HIV is an enveloped virus which causes acquired immune deficiency syndrome in humans. HIV
infects the host T-cells and macrophages and deteriorates the whole immune system. The virus
initiates the infection process by membrane fusion of the viral and host cell by the membrane
protein gp160. Gp160 forms a trimer structure with each gp160 molecule comprising two noncovalently bonded subunit - gp120 and gp41. Gp120 binds to the host cell receptors and
coreceptors, undergoes conformational change and moves away to expose gp41. The fusion
between the viral and host cell membranes is catalyzed by the ectodomain of the gp41 protein.
A large number of antibody binding sites are located in the gp120 and gp41 sequence. Also,
gp120 and gp41 are capable of eliciting neutralizing antibodies in vivo. However, there are a
large number of drawbacks for the neutralization of the HIV – (a) fast mutation of HIV strains to
evade immune response, (b) non-exposure of most of the antibody epitope regions due to
heavy glycosylation, and (c) non-neutralization of a broad range of HIV strains due to mutation.
Due to these difficulties, there is no vaccine for HIV developed yet.
My research work focused on production of large gp41 ectodomain constructs and biophysical
and structural characterization. The N-terminal fusion peptide (FP) and C-terminal membrane
proximal external region (MPER) are critical for fusion and are postulated to bind to the host
cell and HIV membranes, respectively. One significant finding was observation of synergy

between the N-terminal FP, gp41 ectodomain core region, and C-terminal MPER in vesicle
fusion. The oligomeric state of gp41 ectodomain has been studied. Conditions are found with
predominant monomer or hexamer but not trimer and these may be oligomeric states during
fusion. Monomer gp41 ectodomain is hyperthermostable and has helical hairpin conformation.
A new HIV fusion model has been proposed through this work, where: (1) hemifusion of
membranes is catalyzed by folding of gp41 ectodomain monomers into hairpins; and (2)
subsequent fusion steps are catalyzed by assembly into a hexamer with FPs in an antiparallel sheet arrangement.
Another reason of interest was that MPER is a highly conserved region which can bind to few
broadly neutralizing antibodies effective against various strains of HIV. Two of these antibodies
bind to our designed and produced gp41 ectodomain constructs and therefore, adds a step
forward towards investigation of the gp41 ectodomain as an immunogen in HIV vaccine
development.

Copyright by
KOYELI BANERJEE
2014

To my Father, Late Nityananda Pramanik

v

ACKNOWLEDGEMENTS


I would like to thank my advisor, Dr. David Weliky for his enormous support and

guidance in research and life. I would be highly obliged to Dr. Weliky for guiding and
encouraging me through this great journey of learning and making it a splendid experience. I
was fortunate for I was given freedom to explore various techniques in my research project and
learn and share innovative ideas. Dr. Weliky has always been like a pathfinder whenever I felt
directionless in the broad field of research. I would also thank him for teaching me to think
critically and suggestions to interact with the scientific community efficiently. I am grateful to
him for his patience and molding me into a responsible scientist.


I would like to acknowledge members of my guidance committee including Dr. James

Geiger, Dr. Gary Blanchard, and Dr. Daniel Jones. I would like to express my sincerest gratitude
to all my committee members for the helpful discussions and support throughout the whole
journey in the Graduate school.


I am grateful to present and past members of Weliky-group. I would like to thank my

previous group members Dr. Kelly Sackett, Dr. Matthew J. Nethercott, Dr. Scott Schimck, and
Dr. Erica P. Vogel for helping me to adapt in the new lab during my initial days and discussing
with me whenever required. I would like to thank Dr. Erica P. Vogel, Dr. Matthew J. Nethercott
and Dr. Kelly Sackett for teaching me various techniques performed in our lab. I am grateful to
Erica for her patience to train me in different molecular biology and protein biochemistry
techniques. I am also grateful to my present lab members Dr. Charles Gabrys, Li Xie, Punsisi
Upeka Ratnayake, Ujjayini Ghosh, Lihui Jia, Shuang Liang, and Robert Wolfe for being so friendly
and helpful. . It was a great learning experience interacting with Li regarding NMR techniques. I
vi

would also like to extend my appreciation to Punsisi for the helpful discussions regarding
protein purification methods.


My journey in the graduate school would not have been possible without the support

and encouragement of my family; my Mom, in-laws, my sister, uncles and aunts, my husband
and my son. My Mom, Bela Pramanik, has always been very encouraging to shape my career
path. She has always taught me to be organized and make every single day productive. I would
like to thank my sister and her family for enormous support and good wishes. Special thanks go
out to my in laws and my uncles and aunts for their best wishes.


I am grateful to Dr. Anindya Chanda, Satyaki Sengupta, and Dr. Iwan Setiawan as I would

have never survived in graduate school without their kind friendship. Thank you all for the
wonderful memories.


My husband Rahul has been a tremendous source of encouragement and motivation.

He always motivated me to go forward no matter what the difficulty is. He always supported
me in adverse situations and encouraged me to be optimistic. I find short of word to say thank
you to him for supporting my passion for higher education. It has been such a great experience
discussing scientific issues with him and learn to think critically. Together we came to MSU with
our toddler son, Raunak who is now eight years old. My son has been very understanding
always. During the most difficult days of graduate life, he has been such a great source of
rejuvenation. You both will remain the greatest source of inspiration for me.

vii

TABLE OF CONTENTS

LIST OF TABLES

xi

LIST OF FIGURES

xii

KEY TO ABBREVIATIONS

xix

CHAPTER 1: Human Immunodeficiency Virus Membrane Fusion Protein Gp41
1.1. Introduction
1.1.1. Fusion Peptide
1.1.2. N-terminal Helix Region-C-terminal Helix Region
1.1.3. Membrane Proximal External Region
1.1.4. Transmembrane Domain
1.1.5. Cytoplasmic Endodomain
1.2. Possible Mechanism of Membrane Fusion
1.3. Mechanism of Fusion of HIV-1 and Host Cell Membranes Caused by Gp41
1.3.1. Model I
1.3.2. Model II
1.3.3. Caveats of Both the Models
1.4. Stoichiometry of Fusion proteins at the Site of Membrane Fusion
REFERENCES

1
2
4
5
7
12
13
13
22
23
24
25
26
27

CHAPTER 2: Materials and Methods
2.1. Site Directed Mutagenesis
2.2. Transformation
2.3. Expression of Gp41 HP Construct
2.4. Detection of Expression of HM using Solid State NMR of Inclusion Bodies
2.5. Solubilization of HM
2.6. Purification of Gp41 Ectodomain Protein Constructs
2.6.1. HP
2.6.2. HM
2.7. Western Blot Analysis
2.8. Peptide Synthesis of N-terminal Fusion Peptide Region of Gp41 Ectodomain
2.9. Production of Full-length Gp41 Ectodomain Constructs by Native Chemical
Ligation
2.10.
Stabilization of Gp41 Ectodomain Protein Constructs
2.11.
Determination of Secondary Structure of Gp41 Ectodomain Constructs
2.11.1. Circular Dichroism
2.11.2. Solid State Nuclear Magnetic Resonance
2.12.
Determination of Oligomeric State(s) by Size Exclusion Chromatography
2.13.
Antibody Binding Studies by Direct Immunoprecipitation

35
36
37
38
40
43
46
46
46
49
49

viii

51
54
55
55
55
57
59

2.14.
Vesicle Fusion Assays by Fluorescence Spectroscopy
REFERENCES

60
62

CHAPTER 3: Structural and Functional Characterization of Gp41 Ectodomain
3.1. Introduction
3.2. Experimental Procedures
3.2.1. HP and HM Inserts
3.2.2. HP and HM Expression, Solubilization and Purification
3.2.3. Synthesis and Purification of FP-HM
3.2.4. Size Exclusion Chromatography
3.2.5. CD Spectroscopy
3.2.6. Western Blots
3.2.7. Immunoprecipitation
3.2.8. Protein Induced Vesicle Fusion
3.3. Results
3.3.1. High Yield Protein Production
3.3.2. Solubility Only at pH 3 or With 6M GuHCl
3.3.3. Monomer or Hexamer and Not Trimer
3.3.4. Hyperthermostable Hairpin Structure of the Monomer
3.3.5. Hairpin Monomer With MPER is Highly Fusogenic
3.3.6. Synergy of FP, Hairpin, and MPERwith the GP41 Copy Number of a Virion
3.3.7. Folded Hairpin Ectodomain With MPER Binds to bNAbs
3.4. Discussion
REFERENCES

65
66
70
70
72
73
75
75
75
75
76
77
77
80
81
84
88
91
94
97
113

CHAPTER 4: Construction of HIV-1 Gp41 Pre-Hairpin Intermediate
4.1. Specific Aims
4.2. Introduction
4.3. Approach
4.3.1. Mutagenesis of CHR
4.3.2. Verification of Formation of Pre-Hairpin Intermediate
4.4. Materials and Method
4.4.1. Construction of Mutant HP and HM
4.4.2. Experimental Procedure
4.5. Results
4.6. Discussion
4.7. Future Work
REFERENCES

120
121
121
123
123
125
127
127
128
129
135
136
137

CHAPTER 5: Future Work
REFERENCES

140
143

ix

APPENDICES
APPENDIX I: Designing of FP-HP by Native Chemical Ligation
APPENDIX II: Determination of Secondary Structure by Solid State NMR
of Membrane Associated FP of Gp41 Ectodomain Construct FP-HP
APPENDIX III: Study of Binding of Designed HIV Pre-Hairpin
Intermediate and Membrane Fusion Inhibitor C34 Peptide
APPENDIX IV: Sample Preparation of Uniformly Labeled Bacterially
Expressed HIV-1 Gp41 Hairpin Ectodomain
APPENDIX V: Location of Experimental Data Files
REFERENCES

x

145
146
150
154
161
165
168

LIST OF TABLES

Table 2.1. Solubilization of inclusion bodies containing HM in various solubilizing agents 45

xi

LIST OF FIGURES

Figure 1.1. (Left) Infection model of HIV, (Right) Electron microscopy pictures of HIV infection
process, (a) Binding of HIV and host cell, (b) Hemifusion of viral and host cell membrane, (c)
Pore formation, and (d) complete fusion and entry of viral genetic material into the host cell.
3
Figure 1.2. (A) Schematic diagram of the HIV-1 gp41 and (B) gp41 ectodomain amino acid
sequence color coded according to the different domains.
4
Figure 1.3. (A) Six-helix bundle of gp41 comprising NHR and CHR and (B) NHR and CHR showing
heptad repeat amino acid positions as orange and yellow respectively. N36 is the 36 residues in
the NHR and C34 is the 34 residues in the CHR.
7
Figure 1.4. (A) NMR structure of MPER in DPC micelle showing the kink at residue W672-N674.
(B) Crystal structure of monomer NHR-CHR-MPER showing MPER as a continuous helix. The
MPER is from L662-I675. The NHR and CHR are denoted as HR1 and HR2 respectively. 9
Figure 1.5. (A) Model of MPER only (bottom) and MPER bound to 4E10 (top). The residues
W672-W676 shown in blue and N671 and W680 shown in magenta undergoes conformational
change upon binding to the antibody. (B) Model of MPER bound to antibody Z13e1 showing
conformational change of MPER helices with change in ψ values
11
Figure 1.6. Stages of membrane fusion

21

Figure 1.7. Proposed energy profile of membrane fusion caused by HIV-1 gp41 envelope
protein. The maximum energy barrier for stalk formation is calculated as ~25 kcal/mol (Kuzmin,
P. I. et al. PNAS (2001), 98). The energy barrier to reach hemifusion and pore formation is lower
than energy barrier for stalk formation. The energy barrier between stalk and hemifusion stage
is due to activation energy required to allow the lipid molecule acyl chains to move away from
their hydrophobic environment and avoidance of water molecules. After hemifusion, there is
complete fusion between the two membranes which lead to pore formation. In the whole
process H Two membrane = H Fusion pore
22
Figure 1.8. Model I: Schematic diagram of membrane fusion caused by gp41. (1) Trimer
gp120+gp41, (2) dissociation of gp120 from gp41 and exposure of gp41 FP, (3) SHB folding of
gp41 and hemifusion of viral and host cell membrane, and (4) pore formation with close
apposition of FP and TM of gp41. ‘A’ denotes viral membrane and ‘F’ denotes FP region 24
Figure 1.9. Model II. Stages of fusion: (a) gp120+gp41 trimer, (b) gp41 PHI formation with FP
insertion into host membrane, (c) hemifusion caused by PHI, and (d) SHB formation
25
Figure 2.1. Amino acid and DNA sequence of HP construct
xii

38

Figure 2.2. SDS-PAGE gel showing RP-HPLC purified HP in glacial acetic acid at pH 3.0

40

Figure 2.3. SDS-PAGE gel showing IMAC purified HP in the presence of urea at ph 7.4
Figure 2.4. Amino acid and DNA sequence of HM

40
41

Figure 2.5. Chemical shift corresponding to 13CO- Leu

42

Figure 2.6. Solubilization of inclusion bodies in various solubilizing agents

44

Figure 2.7. SDS-PAGE gel showing low solubilization and negligible binding of HM with 2Gly-4His
tag in 8M urea with Co2+ resin
47
Figure 2.8. SDS-PAGE gel showing inefficient binding of HM with 4Gly-4His tag in 3xCMC
Sarkosyl with Co2+ resin
48
Figure 2.9. SDS-PAGE gel showing binding of HM with 2Gly-4His tag in 6M GuHCl with Co2+ resin
and elution with high purity. The bands near 25 kD are dimer bands, confirmed by western blot
with anti-His antibody
48
Figure 2.10. Schematic representation of t-Boc solid phase peptide synthesis

51

Figure 2.11. Mechanism of native chemical ligation. The FP23 that is synthesized by t-Boc SPPS
is shown having a thiol on the C-terminus. To make the reaction more rapid, a catalyst MPAA is
used. MPAA undergoes thioester exchange, and the Hairpin with the N-terminal Cys can easily
form thioester-linked intermediate. Finally the more stable product, i.e., the peptide bond is
formed
53
Figure 2.12. The REDOR pulse sequence

57

Figure 2.13. SEC A280 of MW standards loaded on (A) Superdex 75 and (B) Superdex 200
columns. The protein standards used were (A) Conalbumin (75 kDa), Ovalbumin (44 kDa),
Carbonic Anhydrase (29 kDa), Ribonuclease A (13.7 kDa), and Aprotinin (6.5 kDa) and (B)
Thyroglobulin (669 kDa), Ferritin (440 kDa), Aldolase (158 kDa), Conalbumin (75 kDa),
Ovalbumin (44 kDa), Carbonic Anhydrase (29 kDa), Ribonuclease A (13.7 kDa), and Aprotinin
(6.5 kDa)The Vo is the void volume and corresponds to 2 MDa Blue Dextran. Kav vs log10(MW)
for the (C) Superdex 75 and (D) Superdex 200 columns. The Kav = (Ve – Vo)/(Vc – Vo) where Ve
and Vc are the elution and column volumes, respectively
58
Figure 3.1. (A) Schematic diagram of HIV gp41 where FP  fusion peptide, NHR  N-heptad
repeat; CHR  C-heptad repeat, MPER  membrane-proximal external region, TM 
transmembrane domain, and Endo  endodomain. (B) Amino acid sequences of HP, HM, and
FP-HM. The epitopes of the 2F5 and 4E10 bNAbs are marked
71
Figure 3.2. DNA sequence of the HM insert

71

xiii

Figure 3.3. MALDI mass spectra of (A) the HM and (B) FP-HM peaks from the first HPLC
purification, Fig. 4A. Inset is SDS-PAGE where the middle lane is the FP-HM peak of Fig. 4A and
the right lane is purified HM
74
Figure 3.4. (A) First-round RP-HPLC profile of FP+HM ligation. (B) Second-round RP-HPLC of FPHM peak from panel A
74
Figure 3.5. (A) 13C SSNMR spectra of a cell pellet harvested from centrifugation of a cell lysis in
PBS. The E. coli cells contained a plasmid with the HM insert and expression was induced for
two hours in minimal medium containing 13CO-Leu. Any expressed HM will therefore be 13COLeu labeled. The SSNMR acquisition parameters included a 9.4 T magnetic field, 4 mm diameter
rotor, 8 kHz magic angle spinning frequency, and ~1 day of signal averaging. The ratio of
integrated isotropic 13CO intensity (peaked at ~175 ppm) to integrated aliphatic intensity (0 –
90 ppm region) translates to expression of ~300 mg HM/L culture. (B) SDS-PAGE of purified HM
(MW = 13.7 kDa) and HP (MW = 11.6 kDa). (C) MALDI MS of FP-HM ligation product twicepurified by RP-HPLC. The experimental ratio [m/z(FP-HM):m/z(HM)] is 1.1522 and matches the
ratio of expected MWs, [MW(FP-HM):MW(HM)] = 1.1522
79
Figure 3.6. SEC A280 profiles for HP with (A) 1 mg/mL loading, Superdex 200 column and (B) 10
mg/mL loading, Superdex 75 column. The loading and running buffer was 50 mM sodium
formate pH 3.2, 150 mM NaCl, and 0.2 mM TCEP. The blue numbers are the MW standards in
kDa and their respective elution volumes are the downward blue arrows. There are
accompanying SEC profiles of the MW standards as well as plots of Kav vs log10(MW) (Chapter 2,
Figure 11)
82
Figure 3.7. SEC A280 profiles from a semi-preparative Superdex 200 column for HP and HM in
buffers containing 6 M GuHCl and 150 mM NaCl. The pH 3.2 buffer was 50 mM sodium formate
with 0.2 mM TCEP and the pH 7.4 buffer was 10 mM phosphate with 2 mM DTT. For each run,
the loading and running buffers were the same and the loading stock solution had a 1 mg/mL
protein concentration. The blue numbers are the MW standards in kDa and their respective
elution volumes are the downward blue arrows. The most prominent peaks in the profiles
correspond to hexamer masses and vertical dashed lines show the elution volume offset
between the HP and HM hexamers. Smaller peaks most consistent with dodecamer and 24-mer
masses are identified by shaded regions. For HM, there is also a small peak associated with the
Vo void volume, i.e. aggregates with MW  2 MDa
83
Figure 3.8. Evidence for thermodynamic equilibrium between hexamers and larger oligomers.
SEC with A280 detection was run for HP and HM in buffer containing 50 mM sodium formate at
pH 3.2 with 150 mM NaCl, 6 M GuHCl, and 0.2 mM TCEP (Figure 3.7). The hexamer peak was
collected and loaded onto the Superdex 75 column for SEC with this same buffer. The loading
solution contained (A) 0.3 or (B) 0.2 mg protein/mL. Each blue number is the MW of a standard
in kDa and the blue downward arrows is its elution volume from Figure 2.13, Chapter 2.The
resultant profiles for (A) HP and (B) HM show a mixture of hexamers and larger oligomers
similar to the profiles of the initial run (Figure 3.7). This similarity supports this mixture as the
thermodynamic equilibrium state for the protein
84
xiv

Figure 3.9. (A) CD spectra of HP and HM in pH 3.2 buffer. All spectra show 222 indicative of a
significant fraction of  helical structure. (B) 222 vs temperature for HM at pH 3.2. No unfolding
transition is observed. (C) CD spectra of WT and mutant HP at pH 3.2. For a SHB trimer, the
D632A mutation could disrupt the inter-monomer salt bridge with K574 and the W628A
mutation could disrupt the intra-monomer hydrophobic interaction with W571. More positive
CD signal for the double but not the single mutant is consistent with monomer rather than SHB
trimer HP at pH 3.2. (D) CD spectra of HP and HM in buffer with 6 M GuHCl. The spectra show
222 indicative of a significant fraction of  helical structure. The [protein]  15 M (~0.2
mg/mL), the pH 3.2 buffers contained 50 mM formate with 0.2 mM TCEP, and the pH 7.4
buffers contained 10 mM phosphate and 2 mM DTT
87
Figure 3.10. Vesicle fusion at 37 oC. Protein was added just prior to the increase in fusion with
~5 s assay dead time. For panels A and B, the vesicles were negatively-charged and for panels C
and D, the vesicles were positively-charged. The calculated protein charge is +16, –2, and –4 at
respective pH values 3.2, 7.4, and 9.0. The protein:lipid mole ratios were: (A) 1:700, (B) 1:75,
and (C, D) 1:300, with Chol not included in the lipid quantity. The data overall support the
importance of attractive protein/vesicle attractive energy for fusion at low protein:lipid ratios.
The protein stock contained 20 M protein (~0.25 mg/L) in 10 mM formate at pH 3.2 with 0.2
mM TCEP which are condition for predominant monomeric protein. For panels A and B, [POPC]
= 120 M, [POPG] = 30 M, and [Chol] = 75 M, and for panels C and D, the vesicles contained
[POPC] = 120 M, [DOTAP] = 30 M, and [Chol] = 75 M. Vesicle buffers were 10 mM formate
at pH 3.2, 5 mM HEPES/10 mM MES at pH 7.4, and 10 mM Tris-HCl at pH 9.0
90
Figure 3.11. Vesicle fusion at 37 oC. A 7.5 L aliquot of protein was added just prior to the
increase in fusion and the final total volume was 1200 L. The POPC:POPG:Chol vesicle
composition and buffers were the same as Fig. 6. The protein stock buffer was 10 mM formate
at pH 3.2 with 6 M GuHCl and 0.2 mM TCEP and following addition of protein stock, the assay
buffer contained 40 mM GuHCl. For the pH 3.2 assays, the stock [protein] = 20 M (~0.25
mg/mL), and for the pH 7.4 assays, the stock [protein] = 160 M (~2 mg/mL). The
[protein]:[POPC+POPG] = 1:1200 for the pH 3.2 assays and 1:150 for the pH 7.4 assays 92
Figure 3.12. Dose response of vesicle fusion induced by HM and FP-HM at pH 3.2. The assay
conditions were the same as Fig. 7A except that the protein stock concentrations were 20, 10,
and 5 M for [protein]:[total lipid] = 1:1200, 1:2400, and 1:4800, respectively
93
Figure 3.13. Vesicle fusion induced by GuHCl at (A) pH 3.2 or (B) pH 7.4. A 7.5 L aliquot
containing 6 M GuHCl was added to a vesicle suspension with final total [GuHCl] = 40 mM in a
volume of 1200 L. The assay conditions were the same as Fig. 7 except that there was no
protein in the aliquot that contained GuHCl
93
Figure 3.14. Western blots showing binding of HM to the 4E10 and 2F5 bNAbs and weaker
binding of HP to 2F5. The left-side arrows are MWs in kDa
95

xv

Figure 3.15. SDS-PAGE of the immunoprecipitation of HP and HM with the 4E10 and 2F5 bNAbs.
There is stronger binding of HM to 4E10 and 2F5 and weaker binding of HP to 2F5. The protein
stock buffer contained 50 mM sodium formate at pH 3.2 with 150 mM NaCl. For the right three
lanes of the panel A gel, the buffer also contained 6 M GuHCl whereas for the left three lanes of
panel A, the GuHCl was absent. Binding was done in PBS at pH 7.4 and for the right lanes, the
diluted [GuHCl] = 40 mM. The center lane is the MW ladder in kDa
96
Figure 3.16. SDS-PAGE of the immunoprecipitation of HP and HM with IgG. Protein binding to
IgG is not observed. The protein stock buffer contained 50 mM sodium formate at pH 3.2 with
150 mM NaCl. For the right two lanes of the gel, the buffer also contained 6 M GuHCl. The
antibody binding was done in PBS at pH 7.4 and for the right lanes of the gel, the diluted
[GuHCl] = 40 mM. The inner lanes are MW ladders in kDa
97
Figure 3.17. Working structural models of the gp41 ectodomain with Fig. 1 color coding.
Monomeric HP is modeled as the hairpin structure of a monomer in the SHB. HM and FP-HM
hexamers are modeled as two SHB trimers that contact at their N-/C-terminal interfaces. This
model correlates with the observed intermolecular antiparallel  sheet structure of FPs 100
Figure 3.18. The monomeric elution peak of protein GB1 in PBS in the presence of 6M GuHCl at
(A) pH 3.2 and (B) pH 7.4. The elution volume corresponds to the molecular weight of ~6.3 kDa
and ~9 kDa at pH 3.2 and 7.4 respectively. The size exclusion chromatography was done at
Superdex 75 column. The blue arrows show the elution volumes of MW standards with the
MWs given in kDa
103
Figure 3.19. Model for membrane fusion that includes the gp41 ectodomain monomer and
hexamer. The different regions of gp41 are color coded according to Fig. 1 and the TM and
endodomains are not shown. One of the monomers is not displayed in steps 3-5. The initial
gp41 structure of step 1 is based on the ~5 Å gp140 structures and the final SHB structure of
step 7 is based on high-resolution structures
109
Figure 4.1. HIV fusion model. (a)Native state of envelope protein before fusion, (b) Pre-Hairpin
intermediate of gp41, and (c) Final Hairpin state of gp41
122
Figure 4.2. Helical wheel representation of NHR and CHR. Three NHR helices and one CHR helix
are represented as helical wheel projections. It is an end-on-view of the NHR-CHR complex
looking down the axis of the trimer. The NHR helices interact with other NHR helices at the “a”
and “d” positions (colored orange). Positions of the NHR and the CHR helices that occupy the
interhelical space between two NHR helices and one CHR helix are shaded yellow. The residues
which interact among themselves are colored accordingly
125
Figure 4.3. (Left) Far-UV CD analysis of Hairpin showing 100% helicity with the mean residue
molar ellipticity at 222nm (θ222) is -33,000 deg cm2 dmol-1. For inset, abscissa is the temperature
scale in °C, and the ordinate is θ222, showing the thermal stability of Hairpin with temperature.
(Right) Thermal stability of Hairpin () and N70 (+). The melting temperature of N70 is 45 °C
126
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Figure 4.4. Amino acid sequence of HP protein construct, DNA sequence of HP WT and HP M3
128
Figure 4.5. The SHB trimer of gp41 comprising of inner core NHR and outer CHR. Each monomer
is colored differently. Top, residues shown forms intermonomer salt bridge, and bottom, shows
residues forming hydrophobic interaction between intramonomer strands
130
Figure 4.6. CD spectra of WT and mutant HP at pH 3.2. For a SHB trimer, the D632A mutation
disrupts the inter-monomer salt bridge with K574 and the W628A mutation disrupts the intramonomer hydrophobic interaction with W571. The [protein]  15 M (~0.2 mg/mL), the pH 3.2
buffers contained 50 mM formate with 0.2 mM TCEP
132
Figure 4.7. CD spectra of HP WT and HP M3 at pH 3.2. W631R mutation disrupt the intramonomer hydrophobic interaction with W571. The [protein]  10 M (~0.2 mg/mL), the pH 3.2
buffers contained 50 mM formate with 0.2 mM TCEP
133
Figure 4.8. CD spectra of HM WT and HM M3 at pH 3.2. For a SHB trimer, W631R mutation
along with W628A disrupts the intra-monomer hydrophobic interaction with W571. The
[protein]  10 M (~0.2 mg/mL), the pH 3.2 buffers contained 50 mM formate with 0.2 mM
TCEP
134
Figure 4.9. Estimation of thermal stability of mutant HM. Left, measurement of thermal
unfolding of HM WT and HM M3 with mean residue molar ellipticity as a function of
temperature at 222nm and pH 3.2, and right, CD spectra of thermally denatured protein after
refolding
135
Figure AI-1. SDS-PAGE gel of Hairpin (non His-tag) purified by RP-HPLC. MW of Hairpin (non Histag) is 10.7 kDa. Lanes: 1- Hairpin; 2- Standard
147
Figure AI-2. CD spectra of the refolded FP-HP

149

Figure AI-3. SDS-PAGE gel comparing FP-HP and Hairpin. MW of FP-HP is 12.8kDa, and Hairpin
(non His-tag) is 10.7kDa. Lanes: 1- Standard, 2- FP-HP, 3, 4 –Hairpin
149
Figure AII-1. (Left) The structure of part of the FP showing the positions of the 13C and the 15N
labels on Leu (L7) and Phe (F8) respectively. (Right) REDOR subtracted signal for L7F8 in
DTPC:DTPG:Chol vesicles. The CO peak of L7 was referenced to the methylene peak of
adamantane at 40.5 ppm. The chemical shifts of 178.5 and 173.7 ppm correspond to α-helix
and β-sheet respectively
152
Figure AIII-1. (A) Schematic diagram of gp41 showing different domains – fusion peptide (fp),
NHR (Heptad repeat 1), CHR (Heptad repeat 2), transmembbane (tm) and endodomain (cyto).
The C34 peptide is shown to compare the sequence length with CHR. The three residues that
bind into the groove are shown in red. (B) Down the axis view of SHB structure of gp41
ectodomain, (C) sidewise view of SHB showing the position of CHR on the NHR core grooves. (D)

xvii

The packing of the C34 on the N36 (NHR) grooves. The N36 is represented as a molecular
surface and the three residues of C34 which fit into the binding pocket are shown in red 155
Figure AIII-2. Circular dichroism spectra of (A) 5μM of HM M3 and (B) 5μM C34 at pH 3.0
158
Figure AIII-3. Secondary structure of HM M3 + C34, 5 μM each. The incubation was performed
overnight at 4°C. The CD was performed at pH 3.0
159
Figure AIII-4. The secondary structure HM M3 + C34, 5 μM each. The protein was heated for 2
min and the C34 was added and incubated overnight at 4°C
159
Figure AIV-1. MAS cross polarization signal for 13C labeled inclusion body containing HP protein.
The CO peak of HP was referenced to the methylene peak of adamantane at 40.5 ppm 164

xviii

KEY TO ABBREVIATIONS

AUC

analytical ultracentrifugation

BCA

bicinchoninic assay

bNAb

broadly neutralizing antibody

CD

circular dichroism

Chol

cholesterol

CHR

C-terminal helix region

DOTAP

1,2-dioleoyl-3-trimethylammonium-propane (chloride salt)

FP

fusion peptide

gp160

glycoprotein 160

gp140

glycoprotein 140

gp120

glycoprotein 120

gp41

glycoprotein 41

GuHCl

guanidine hydrochloride

IB

inclusion body

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HIV

Human immune deficiency virus

IPTG

isopropyl β-D-1-thiogalactopyranoside

LB

Luria-Bertani

MES

2-(N-morpholino)ethanesulfonic acid

MPAA

S-Trityl-β-mercaptopropionic acid

MPER

membrane-proximal external region

SHB

six-helix bundle
xix

NHR

N-terminal helix region

N-NBD-DPPE N-(7-nitro-2,1,3-benzoxadiazol-4-yl) (ammonium salt) dipalmitoylphosphatidylethanolamine
N-Rh-DPPE

N-(lissamine rhodamine B sulfonyl) (ammonium salt) dipalmitoylphosphatidylethanolamine

PHI

pre-hairpin intermediate

POPC

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

POPG

1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt)

RP

recombinant protein

RP-HPLC

reversed-phase HPLC

SEC

size-exclusion chromatography

SSNMR

solid-state nuclear magnetic resonance

TCEP

Tris(2-carboxyethyl) phosphine hydrochloride

xx

CHAPTER 1
Human Immunodeficiency Virus Membrane Fusion Protein Gp41

1

1.1.

Introduction:

Acquired Immune Deficiency Syndrome (AIDS) is a disease of the human immune system
caused by the Human Immunodeficiency Virus (HIV). According to World Health Organization,
there were 1.1 million people living with HIV till the year 2012 in the United States and 1.6
million annual deaths worldwide.
The viral entry to the host cell is postulated to occur in two ways. (1) Fusion of the viral and the
host cell membrane and then release of the viral genetic material into the host cell which
eventually integrates with the host genome (1,2). (2) The second is the endocytic pathway. The
virus enters the cell cytoplasm through the endosomes and then complete fusion occurs
followed by delivery of viral genetic material into the cell cytoplasm (3,4). The mechanism of
viral entry through fusion of cell membrane is believed to be more prevalent based on the
evidence that there is shorter lag between viral membrane protein-host cell receptor
coreceptor complex and cell-cell fusion (5) compared to delayed endosomal fusion of HIV (3).
The process of membrane fusion is mediated by HIV membrane glycoprotein gp160. Gp160 is
comprised of non-covalently associated outer receptor binding subunit, gp120 and an inner
fusion protein, gp41(1). The membrane spanning gp41 participates in the fusion of the viral
membrane with the host cell membrane and this membrane fusion creates a pore which can
transfer the viral genetic material into the host cell (Figure 1.1).

2

Figure 1.1. (Left) Infection model of HIV, (Right) Electron microscopy pictures of HIV infection
process, (a) Binding of HIV and host cell, (b) Hemifusion of viral and host cell membrane, (c)
Pore formation, and (d) complete fusion and entry of viral genetic material into the host cell(6).
The gp160 in HIV membrane is activated for fusion by the interactions with CD4 (receptor) and
CXCR4 or CCR5 (coreceptor) of the T-cell or macrophage cell membrane. Gp120 binds to the
CD4 receptor and coreceptors. The binding results in a conformational change in gp120 and as
a result gp120 dissociates with gp41 exposed(1). The gp41 plays a significant role in fusion
between viral and host cell membranes. Gp41 consists of different regions such as ectodomain
with amino acid residues 512-683, comprising of N-terminal fusion peptide (FP), an N-terminal
helix region (NHR), an immunodominant loop region, a C-terminal helix region (CHR),
membrane proximal ectodomain region (MPER), the transmembrane region (TM) amino acid
residues 684-705, and the endodomain (cytoplasmic tail) amino acid residues 705-856 (Figure
1.2). Each region of gp41 plays a unique role.

3

Figure 1.2. (A) Schematic diagram of the HIV-1 gp41 and (B) gp41 ectodomain amino acid
sequence color coded according to the different domains.
1.1.1. Fusion Peptide
The fusion peptide (FP) region is a Gly rich hydrophobic N-terminal domain of gp41. There is no
atomic resolution crystal structure of the FP region; however, some NMR structural studies
have been done with FP associated with detergent micelles. The FP is unstructured in solution
whereas it forms α-helical structure in the presence of detergents SDS or DPC (7-9). There have
been contradictory structural studies showing FP in physiologically relevant membrane
environment as β-sheet structure (10,11), whereas, another study shows that FP region
acquires α-helical monomeric structure and β-sheet structure at low and high peptide:lipid
concentration respectively (12). Difference in conformation of FP has been observed with the
presence or absence of cholesterol in the lipid membrane. The FP adopts α-helical structure in
the absence of cholesterol and β-sheet structure in the presence of cholesterol (11). Studies on
FP domain indicate that oligomerization of FP may be important for fusion activity. Lipid mixing
assay and analytical ultracentrifugation

studies of FPmonomer, FPdimer, and FPtrimer shows

maximum fusion activity for the trimer FP construct (13). Mutation of the Val-2 to Glu-2 (V2E) in

4

the N-terminal end shows inhibition of fusion activity and syncytia formation. The effect of V2E
mutation is such that even in the presence of excess wild type gp160, the membrane fusion and
HIV infection is inhibited revealing the functional oligomeric property of FP domain (14). These
studies suggest that oligomerization of FP is essential for fusion of viral and host cell
membrane. The study of interaction of synthetic FP and PG LUVs has been done by lipid mixing
assays, vesicle binding and leakage experiments. The studies suggest that wild type FP can
penetrate through vesicle monolayer and cause permeabilization (12). Similar study performed
with mutant V2E FP peptides showed no destabilization of PG vesicles (15). Also solid state
NMR studies of FP in the lipid membranes show maximum membrane insertion depth and
fusogenicity of trimer FP than compared to monomers or mutated (V2E) FP (16). Based on
these studies, it is could be proposed that probably FP inserts into the host membrane during
the fusion process.
1.1.2. N-terminal Helix Region-C-terminal Helix Region
The high resolution crystal structure of the NHR and CHR without the loop region shows a
highly helical trimer structure forming a hairpin conformation (17,18). This trimer NHR-CHR
structure also known as six-helix bundle (SHB) is proposed to be the structure of gp41 core
region after the fusion process is complete (Figure 1.3A). At the center of the SHB is parallel
trimer NHRs held together by hydrophobic interactions primarily between the Ile or Leu
residues. The amino acid sequence of N-helix region (NHR) and the C-helix region (CHR)
comprise of 4-3 heptad repeats of hydrophobic residues. Heptad repeats are formed by repeats
of hydrophobic residues in the 1st and 4th residue in each seven residue helical turn. The

5

hydrophobic residues in the ‘a’ and ‘d’ positions form the hydrophobic interactions (Figure
1.3B). The CHR helices pack on the outside grooves of the NHR trimer core in antiparallel
orientation. The interaction between the NHR and CHR is predominantly hydrophobic between
the ‘e’ and ‘g’ residues of NHR helices and ‘a’ and ‘d’ residues of CHR helices respectively (figure
3B). Some of the hydrophobic interactions include: (a) W628, W631 of CHR interacts with W571
of NHR, (b) I635 of CHR interacts with L565 and L568 of NHR, and (c) Q653 of CHR has
intramolecular hydrogen bond with Q551 of NHR and Q653 forms intermolecular hydrogen
bond with the backbone CO oxygen of V549 of NHR. There is a salt bridge between the NHR and
CHR between the K574 and D632. The NHR-CHR SHB is very thermally stable with melting
temperatures of ~70o C for shorter ectodomain constructs with hairpin structure whereas
temperatures up to 110o C are observed for longer constructs (19,20). This thermostability has
supported the SHB as the final gp41 structure during fusion. Very recent solution NMR
structural study of NHR-CHR in DPC micelles suggests monomeric structure with very less
interaction between the NHR and CHR. The 13Cα chemical shifts of all the residues of NHR-CHR
construct was compared with the chemical shifts of the residues of the individual peptides NHR
and CHR. It was observed that in the

13 α

C chemical shift pattern of the NHR-CHR construct

exactly overlapped with the individual NHR and CHR peptides. The same group conducted the
study to understand the interaction of the NHR-CHR protein construct with detergent
molecules by paramagnetic relaxation enhancement. It was observed that both the NHR and
CHR helices are amphipathic with the hydrophobic residues lying on one side of the helix and
the hydrophilic residues on the other side of each helix. Based on this observation, it is
proposed that both NHR and CHR can be embedded at the lipid-water interface (21).
6

A

B

NHR

CHR

Figure 1.3. (A) Six-helix bundle of gp41 comprising NHR and CHR and (B) NHR and CHR showing
heptad repeat amino acid positions as orange and yellow respectively (22). N36 is the 36
residues in the NHR and C34 is the 34 residues in the CHR.
1.1.3. Membrane Proximal External Region
Membrane proximal external region (MPER) is the last ectodomain region following CHR
towards the C-terminal end comprising of the amino acids from 662 – 683.The two primary
characteristics of the MPER due to which this is a region of great interest are: the amino acid
residues are highly conserved (23-25), and the region comprise of epitopes for three broadly
neutralizing antibodies (23,25). Broadly neutralizing antibodies are the antibodies which can
stop infection caused by a broad range of HIV strains. HIV-1 can easily mutate to evade the
immune response of the host. The immune response of the host is further non-functional due
to non-exposure of neutralizing epitopes. The gp120 and gp41 can elicit neutralizing antibodies,

7

however, these antibodies are isolate specific (26). Very few broadly neutralizing antibodies are
elicited by the epitopes contained in the highly conserved MPER domain. The broadly
neutralizing antibodies 4E10, 2F5, and Z13 recognize epitopes in the MPER (23,25). Even though
the broadly neutralizing antibodies are efficient enough to inhibit the fusion process by binding
to the MPER epitopes, still there is no success by injecting the antigen into the host body to
elicit antibody (26). Efforts to produce broadly neutralizing antibodies to the conserved
epitopes of MPER proved to be of not much use. The conserved neutralizing epitopes have
been observed to be poor immunogens as they mimic host antigens leading to depletion of
immune tolerance (27).
Structural characterization of MPER peptide in lipid and detergent environment by EPR and
NMR respectively shows kinked helical structure (Figure 1.4A). The kinked structure is
estimated by measurement of immersion depth between specific residues in the MPER and
spin labeled lipid molecules using EPR technique (28).

8

Figure 1.4. (A) NMR structure of MPER in DPC micelle showing the kink at residue W672-N674
(28). (B) Crystal structure of monomer NHR-CHR-MPER showing MPER as a continuous helix
(29). The MPER is from L662-I675. The NHR and CHR are denoted as HR1 and HR2 respectively.
Recent high resolution crystal structure of MPER along with NHR-CHR shows helical structure
(Figure 1.4B) (29). The kinked MPER peptide structure where the kink starts at W672, and the
NHR-CHR-MPER crystal structure containing residue 662LELDKWASLWNWFDI675 shows complete
helical structure with absence of a kink (29,30). X-ray crystal structure study of MPER when
bound to broadly neutralizing antibodies 2F5 show that the N-terminal end

661

LELDKWAS668

within the MPER domain is in helical conformation (31). EPR and NMR structural studies show
change in conformation of the MPER after binding to the antibodies. By EPR studies, the
membrane insertion depth is determined for spin labeled MPER bound to 4E10 antibody with
respect to POPC/POPG membranes. The difference in chemical shifts of amides of the residues
of MPER in bound and unbound state suggests conformational change after binding to
antibodies. Also NMR cross-saturation transfer experiments
9

in which magnetization is

transferred from 1H of methyl groups of the bound antibodies to the amides of residues of perdeuterated MPER further confirms the conformational change of the residues W672, F673,
N674, I675, T676, N671, N677, L679, Y681, I682, and K683 of MPER in contact with the
antibodies (28,30). The kink between W672-K683 changes to a continuous helical structure
upon binding to antibody 4E10 (Figure 1.5A) as observed in the crystal structure of NHR-CHRMPER (29). Upon Z13e1 binding, the dihedral angles of the MPER comprising L669-W672, and
I675-Y681 changes significantly (Figure 1.5B) compared to dihedral angles of MPER in micelle
bound structure in the absence of Z13e1. The backbone dihedral angles φ and ψ of the
residues for the helix region comprising of L669-W672 and I675-Y681 changes from typical αhelical values of -57° and -47° upon binding to Z13e1 . The change in ψ values of the residues
L669-W672 is such that these residues adopt β-strand structure when bound to the antibody
(30).

10

Figure 1.5. (A) Model of MPER only (bottom) and MPER bound to 4E10 (top). The residues
W672-W676 shown in blue and N671 and W680 shown in magenta undergoes conformational
change upon binding to the antibody (28). (B) Model of MPER bound to antibody Z13e1
showing conformational change of MPER helices with change in ψ values (30).
MPER is a Trp rich region. The mutation of the Trp residues reduces the fusogenic activity by
affecting the cell-cell fusion (24). The synthetic peptide comprising part of CHR and MPER
(amino acids 638-673) also known as T-20 or enfuvirtide inhibits syntitia formation and inhibits
fusogenic activity of gp41 in the early stages of viral life cycle (32). Viral life cycle is comprised
of two stages – (1) early stage comprise of viral attachment, transfer of viral genetic material
(RNA) to the host cell, reverse transcription of viral RNA to viral DNA in the host cell cytoplasm,
insertion of viral DNA to host nucleus, and integration of viral DNA to host cell DNA in the host
nucleus and (2) late stage is comprised of transcription, translation, assembly of viral proteins
and genetic material, budding, maturation, and new virus formation or synctitia formation with
neighboring host cells.

11

As enfuvirtide is a peptide mimic of the CHR+MPER domain, it is proposed that enfuvirtide
binds to NHR in the early steps of gp41 conformational change during membrane fusion (33).
The binding of the broadly neutralizing antibodies 2F5, 4E10, and Z13 to the MPER epitopes
reduces the Env mediated fusion activity (25,34,35). Therefore, binding of the antibodies or
peptide enfuvirtide inhibits the fusion of viral and host cell membrane, thus blocking the very
first step of viral life cycle. Studies of MPER and membrane interaction revealed membrane
permeability and fusion (36-38). FP and MPER peptides interact and act synergistically to
enhance membrane fusion (35,36,39,40).
1.1.4. Transmembrane Domain
The transmembrane (TM) domain anchors the gp41 in the viral membrane and comprises 22
amino acids. There is no structural detail of the TM region only, however, according to
computer simulations data, it acquires α-helical structure (41) and has tendency to form a
trimer (42). The effect of highly conserved TM in the viral membrane fusion has been studied
which gives contradictory result. The mutations in the amino acid residues between 696-707
showed loss in fusion activity while the anchoring of the Env in the lipid bilayers remained same
(43). Also substitution of the TM domain with C-terminal glycosyl- phosphatidylinositol lipid
anchor reduced the fusion activity considerably (44,45). On the contrary, another study of
fusion activity and infectivity by complete substitution of gp41 TM by cytoplasmic 180 residues
at the C—terminal from cellular protein CD22 showed no change in function (46). Future
studies on the role in fusion activity by TM domain would be interesting.

12

1.1.5. Cytoplasmic Endodomain
The gp41 cytoplasmic endodomain (Endo) is the C-terminal region after the TM domain and
comprise of ~150 amino acids. To date there is no high resolution structural information of the
cytoplasmic Endo domain. According to the computational modeled structure, gp41 Endo is a
single-pass membrane protein (41). However, another monoclonal antibody binding study
shows that Endo has at least one membrane spanning domain possibly exposing the epitope on
the surface of the virion (47). The study of HIV-1 particle entry during virus-cell fusion by
fluorescence assay showed immature virus particles were less active than mature virus
particles. The inactivity of the immature virus particles is attributed to interaction of
cytoplasmic Endo with matrix protein, and only dissociation of Endo from matrix protein and
nuleocapsid protein leads to gp41-mediated fusion (48). Functional studies of the cytoplasmic
endodomain by mutation reveal that it plays a significant role in incorporation of Env protein
into the viral membrane (49,50). Interaction of cytoplasmic endodomain with viral matrix
protein has been suggested by mutagenesis of the C-terminal Endo (51,52).
1.2.

Possible Mechanism of Membrane Fusion

Stages of Membrane Fusion – Fusion of two membranes is a crucial step for viral entry into host
cells. Membrane fusion proceeds through the following steps: (1) contact between outer leaflet
membrane surface, (2) stalk formation, (3) hemifusion – fusion of outer leaflet of the two
membranes, and (4) pore formation (Figure 1.4). The whole process of membrane fusion is an
isothermal process (Figure 1.7). X-ray measurements have estimated the distance between
biologically relevant membranes as 2-3 nm (53). Membranes with no net charge on the lipid
13

molecules are at equilibrium due to two interactions – Van der Waals attraction (long range)
and repulsive interactions (short range). When the distance between the membranes is <3 nm,
energy is required to overcome the repulsive force between the membrane outer leaflets of the
two membranes (54). This repulsive force is also known as hydration force. In this membrane
environment, the water molecules tend to bind to the polar head groups of the lipid molecules
by hydrophilic interaction. For a membrane fusion between viral and host cell to occur, energy
is required to displace the bound water molecules, also known as desolvation energy. The
desolvation energy is reduced by contact between the two membranes comprising of a small
area compared to flat membrane surfaces. This contact at a small area is known as stalk (Figure
1.6B). After the stalk formation, there is the stage which tends to rearrange the lipid molecules
of the inner leaflet (Figure 1.6C). Energy is required to allow the lipid molecule acyl chains to
move away from their hydrophobic environment and avoidance of water molecules, during the
initial stages of stalk formation (Figure 1.6C and 1.7). This stage is followed by hemifusion stage
(Figure 1.6D). In the hemifusion stage, the hydrophobic lipid acyl chains rearrange themselves
by not getting exposed to water surface (54,55). The energy of the stalk formation is higher
than hemifusion stage because more energy is required for the desolvation penalty to be paid
during stalk formation (Figure 1.7). The calculated (HStalk - HTwo Membrane ) = ~25 kcal/mol (54,56).
Hemifusion stage is followed by pore formation (Figure 1.6E). Calorimetric experimental studies
of gp41 comprising NHR-CHR shows H unfolding = ~65 kcal/mol (57). It could be that the energy
required for all the stages of membrane fusion is being supplied by the energy released during
the conformational changes in the viral membrane protein. To date there is no experimental
evidence of this energy exchange between membranes and gp41 protein folding, however, it
14

can be proposed that the folding of gp41 NHR-CHR ectodomain may be an exothermic process
releasing ~65 kcal/mol of energy and that energy is utilized to overcome the repulsive force
during membrane fusion. However, this hypothesis of membrane fusion caused by protein
folding is not consistent with our observation of fusogenicity of already folded hairpin
conformation of gp41 ectodomain. Gp41 ectodomain core sequence comprising of NHR-short
loop-CHR (HP) forms stable folded hairpin conformation at pH 3.2 and also causes vesicle
fusion. Therefore, it is hypothesized that the binding of the protein molecules to the lipid
membrane perturbs the lipid molecules increasing the kinetic energy thereby causing local
heating in the membrane. The amount of local heating is calculated as follows:
Using the equation H = mcT ……….(1)

where H is Heat required
m is mass
c is specific heat
T is change in temperature

There is electrostatic interaction between the protein molecules and lipid molecules in
membrane. Considering each protein molecule as a cylinder, then the number of lipid
molecules in contact is calculated as
For Hairpin protein, r = 6 Å, and h = 50 Å,
Surface area of each protein molecule = 2πr(r+h) = 2112 Å2

15

Possibly half of the protein molecule surface will be in contact with the lipid molecules. Area of
each lipid headgroup is ~70 Å2.
Therefore, number of lipid molecules in contact with protein molecule = 1056 Å2/70 Å2 = 15
Energy associated with the perturbation of 15 lipid molecules is
E = (ke z1 z2)/εr ……….(2)

where z = charge on molecules
r = distant between lipid and protein molecules
ke = Coulomb’s constant
ε = relative permittivity of lipid membrane

However, for ionic interaction to occur between the protein molecules and lipid molecules, we
need to estimate the electrostatic energy between the charged molecules only. Only 20% of the
lipid molecules in vesicles (according to our sample preparation) are negatively-charged at
physiologic pH, therefore, from 15 lipid molecules only 3 will efficiently cause electrostatic
interaction with charged protein molecules.
The charge of HP or HM is +2 at physiologic pH.
We are considering the protein and lipid interaction on the outer surface of the membrane
leaflet, we therefore, consider the dielectric constant near the lipid head groups, which is ~30
(58).
E = [(8.968x 109 J m/C2) x (1.602x 10-19)2 C2 x 3 x 2]/30 x 5x 10-10 m
16

E = 1 x 10-19 J ……….(3)
This energy eq. (3) is being distributed among 15 lipid molecules as the protein molecule is in
contact with the same number of lipid molecules.
The volume of each lipid molecule is calculated by considering each lipid molecule as a cylinder,
and radius as 2.4 Å. Therefore, Volume lipid = 4.5 cm3……….(4)
Substituting values (3) and (4) in eqn. (1) we get:
T = (1 x 10-19 J)/ {[(4.5 x 1022 g) x 15] x [(4.2 J/K.g)]}= 3.6 K ~ 4 K ……….(5)
Thus, there could be increase in temperature by ~ 4 K in the lipid membrane due to interaction
with one protein molecule.
Considering the exothermic energy due to folding of HP as ~ 65 kcal/mol, we can calculate the
local heating in the lipid membrane each protein molecule could cause.
E = 65 kcal/mol = 4.5 x 10-19 J………. (6)
Substituting the value from eq (6) in eq (1), we get
T = (4.5 x 10-19 J)/ {[(4.5 x 1022 g) x 15] x [(4.2 J/K.g)]} = 15.9 K ~ 16 K ……….(7)
Therefore, from equations (5) and (7) we observe that the difference in temperature due to
local heating caused by membrane perturbation by each protein molecule is 4 times less
compared to the difference in temperature that could be caused by the energy released by
folding of each protein molecule.
17

Due to this increase in the local temperature, the reaction rate increases (equation 6.) favoring
the forward reaction towards membrane fusion.
According to Arrhenius equation:
k = Ae-Ea/RT

……….. (8)

where k is rate constant
E(A) is Activation energy
R is gas constant
T is Temperature in Kelvin
A is frequency factor constant

lnk = lnA – (Ea/RT) ……….(9)
For temperatures T1 (initial) and T2 (final), the reaction rates would be k1 and k2.
Equation (9) becomes:
ln (k2/k1) = (Ea/R)[(1/T1) – (1/T2) ……....(10)
Considering the activation energy for stalk formation during membrane fusion as ~ 25 kcal/mol
(56) we have:
Ea = 25 kcal/mol = 1.7 x 10-19 J
T1 = 310 K
(I) Considering the T = 4 K , (from eq. 4)

18

T2 = 314 K
R = 8.314 J/K mol
Substituting all the values in eq. (10) we get
ln (k2/k1) = (1.7 x 10-19 J /8.314 J/K mol)[(1/310 K) – (1/314 K)
ln (k2/k1) = [(1.7 x 10-19 J /(8.314 J/K mol)x(mol/6.022 x 1023)][(1/310 K) – (1/314 K)]
ln (k2/k1) = 0.51
k2/k1 = 1.7 ≈ 2
or, k2 = 2k1 ..........(11)
Therefore, the reaction rate for membrane stalk formation doubles due to local heating caused
by perturbation of lipid molecules by folded hairpin gp41 ectodomain.
(II) Now considering T = 16 K , (from eq. 7)
T2 = 326 K
R = 8.314 J/K mol
Substituting all the values in eq. (10) we get
ln (k2/k1) = (1.7 x 10-19 J /8.314 J/K mol)[(1/310 K) – (1/326 K)
ln (k2/k1) = [(1.7 x 10-19 J /(8.314 J/K mol)x(mol/6.022 x 1023)][(1/310 K) – (1/326 K)]
ln (k2/k1) = 1.95
19

k2/k1 = 7
or, k2 = 7k1 ……….(12)
Therefore, the rate after local heating increases by 7 times the initial rate of membrane fusion.

20

Figure 1.6. Stages of membrane fusion.
21

Figure 1.7. Proposed energy profile of membrane fusion caused by HIV-1 gp41 envelope
protein. The maximum energy barrier for stalk formation is calculated as ~25 kcal/mol (Kuzmin,
P. I. et al. PNAS (2001), 98). The energy barrier to reach hemifusion and pore formation is lower
than energy barrier for stalk formation. The energy barrier between stalk and hemifusion stage
is due to activation energy required to allow the lipid molecule acyl chains to move away from
their hydrophobic environment and avoidance of water molecules. After hemifusion, there is
complete fusion between the two membranes which lead to pore formation. In the whole
process H Two membrane = H Fusion pore.
1.3.

Mechanism of Fusion of HIV-1 and Host Cell Membranes Caused by Gp41

HIV-1 and host cell membrane fuse to enable the transfer of viral genetic material into the host
cell and further use the host cell machinery to complete the life cycle and further propagation
(59). The Env glycoprotein i.e. gp160 plays a significant role to mediate the fusion between the

22

two membranes. Gp160 comprise of non-covalently associated gp120 and gp41. Gp120 has
strong affinity for CD4 receptors on T-cells or macrophages (60) and co-receptors CXCR4/CCR5
(1). After binding, gp120 undergoes conformational change and dissociates to fully expose
fusion efficient gp41 (61). Recent cryoelectron tomography study of virion particles with Env
bound to HIV neutralizing proteins showed conformational change of gp120 still attached to
gp41 (62). Another cryoelectron tomography study of conformational change of gp120 upon
binding with CD4 receptor and coreceptor ligand 17b showed gp140 (gp120+gp41 ectodomain
truncated) intact structure (63). However, this gp140 structure is stabilized by disulfide bonds
between gp120 and gp41 residues. Therefore, to date there is no clear structural evidence
whether gp120 completely dissociates from gp41. Based on biochemical assays (61) it is
proposed that after dissociation, gp41 plays the main role in the occurrence of fusion between
the viral and host cell membrane. There are two proposed models for the fusion of membrane
caused by gp41.
1.3.1. Model I
After the dissociation of gp120, gp41 undergoes a conformational change with exposure of FP
towards the host membrane (Figure 1.8 in brackets). In this elongated intermediate stage, the
gp41s are as a trimer bundle. This is followed by formation of SHB of the NHR and CHR with
close apposition of N-terminal FP and C-terminal TM regions which consequently brings the two
membranes close to each other. Viral and host cell membrane hemifusion followed by
complete fusion, and the gp41 FP and TM now lie in the same membrane (Figure 1.6) (18). This
model of membrane fusion caused by gp41 satisfies the energy requirements for the fusion

23

process to take place by the energy released due to folding of gp41 from intermediate state to
final SHB folded state.

Figure 1.8. Model I: Schematic diagram of membrane fusion caused by gp41 (64). (1) Trimer
gp120+gp41, (2) dissociation of gp120 from gp41 and exposure of gp41 FP, (3) SHB folding of
gp41 and hemifusion of viral and host cell membrane, and (4) pore formation with close
apposition of FP and TM of gp41. ‘A’ denotes viral membrane and ‘F’ denotes FP region.
1.3.2. Model II
According to this model, after the gp120 moves away from gp41, the hemifusion of membrane
outer leaflet starts caused by membrane insertion of FP in PHI state. The hemifusion state is
followed by fusion pore formation by folding of PHI to SHB (Figure 1.9) (65). The experimental
evidence supporting the pore formation before SHB formation is shown by closing of pores in
cell/cell fusion assays after the addition of NHR or CHR peptides (66). Experimental study of
vesicle fusion assay of PHI like model at physiological pH and SHB show rapid and negligible
fusion respectively (67).
24

Figure 1.9. Model II. Stages of fusion: (a) gp120+gp41 trimer, (b) gp41 PHI formation with FP
insertion into host membrane, (c) hemifusion caused by PHI, and (d) SHB formation (65).
1.3.3. Caveats of Both the Models
(1) In model I, the sequence of the stages of the protein folding is not consistent with the
experimental observations of inhibition of fusion and synctitia formation by binding of NHR or
CHR peptides (66) or CHR+MPER peptide (32). (2) In both the models I and II, it has been
presumed that the PHI is a trimer structure with NHR forming the core. There is no structural
information of the PHI state of gp41. Recent x-ray crystal and cryo-EM structure of gp140

25

(gp120+gp41 without the FP and MPER) presumably is the state prior to membrane fusion
show loosely packed NHR domains. The gp140 trimer structure could be stabilized by disulfide
bonds between gp120 and gp41 domains and I559P mutation in the NHR to keep the helices
intact (68-71).
1.4.

Stoichiometry of Fusion Proteins at the Site of Membrane Fusion

Structural and functional studies of gp120 and gp41 have led to understand the most probable
fusion mechanism of viral and host cell membrane fusion. Based on fusion mechanism studies,
a number of antiretroviral drugs are being used for the prevention of HIV infection. However,
HIV strains mutate and eventually become resistant against these drugs. Mathematical
estimation of number of trimers per virion concludes that there are 5-8 functional trimers per
virion that effectively participate for viral entry (72,73). Cryoelectron microscopy tomography
study on wild-type HIV-1 virions show ~14 Env protein spikes clustered on the viral membrane
surface (74). Experimental studies on number of Env trimers which may be responsible for
anchoring or binding of the viral Env to the host receptors have been done by cryo-electron
tomography. According to cryo-electron tomography study, the Env trimers form a claw like
structure when the HIV-1 particles interact with CD-4 positive cells. There are ~5-7 Env trimers
which come in contact with target host cells (75). Recent solid state NMR studies of membrane
associated FP in gp41 ectodomain FP-HP shows antiparallel β-sheet arrangement. It is proposed
that this antiparallel arrangement is possible by interleaved FP strands of two gp41 trimers (65).

26

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34

CHAPTER 2
Materials and Methods

35

2.1.

Site Directed Mutagenesis

Our aim was to design the gp41 Hairpin protein with the MPER included. For the addition of the
MPER residues, insertion site directed mutagenesis was performed. Insertion site directed
mutagenesis is initiated with a short primer which contains the required mutation. Primers
were designed complementary to the template DNA sequence near the insertion site to enable
hybridization of the primer to the gene of interest. Two primers i.e. forward and reverse were
designed complementary to the gene of interest DNA double strand. The primers with the
added nucleotides for insertion mutation elongate using DNA polymerase. The amplification of
the required DNA was performed by polymerase chain reaction (PCR).
The various steps of PCR comprised of;
1. Initialization – The initialization is done by a hot start at ~95 °C for 5 min. This is done to
activate the thermally stable Pfu DNA polymerase.
2. Denaturation – This short step of heating (~95 °C) is done for DNA melting of the
template DNA of the template DNA by breaking the H-bonds between the nucleotides of
the double strand DNA. In this step single strand template DNA is obtained.
3. Annealing – In this step the primers anneal with the single strand DNA. To encourage
binding of the primers to the template, the temperature is lowered, typically ~5 °C of
the melting temperature of the primers. It was taken care that the primers were
designed to be GC rich to enable better binding to the template DNA. After primertemplate DNA hybrid formation, synthesis of the whole DNA starts.

36

4. Elongation – After binding of the primer to the template DNA, the DNA polymerase
elongates the DNA complementary to the template DNA by adding dNTPs. The optimum
temperature is typically 72 °C.
5. Final Elongation – This step is also done at 72 °C to make sure that all the single
stranded DNA are completely synthesized.
While performing insertion mutation, various annealing temperatures were applied during
PCR. Additions of 3, 6, or 9 nucleotides could be done at one time. To verify whether the
desired DNA has been produced or not, the final PCR product is run on agarose gel
(electrophoresis) and compared with the DNA ladder.
2.2.

Transformation

The PCR produced plasmid with the insertion of nucleotides was allowed to be taken up by
artificially produced competent BL21(DE3) cells. The plasmid includes the Ampicillin resistant
gene, the lac operon, and the gene which encodes gp41 HP with MPER. After the
transformation, the cells were allowed to grow on agar plate. After overnight growth at 37 °C,
typically one colony was grown in LB. Thus, several 100 mL flasks of LB were inoculated with
single colony and grown overnight at 37 °C. Then the cells were centrifuged and plasmid
extraction was done. The extracted plasmids were the sent for DNA sequencing to verify the
mutation occurrence.

37

2.3.

Expression of Gp41 Ectodomain HP Construct

The gp41 HP plasmid comprised of NHR, short engineered loop, CHR, and a 6-His tag. The insert
was in the pGEM-t vector and Figure 2.1 displays the amino acid sequence with the DNA
sequence. The amino acid sequences are from the HXB2 laboratory strain of the virus and are
described using Env residue numbering. HP includes the NHR residues 535(M535C)-581, a nonnative loop (SGGRGG), followed by CHR residues 628-666 and a non-native H6 tag.
CTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILSG
GRGGWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWHHHHH
H
atg tgc acg ctg acg gta cag gcc aga caa tta ttg tct ggt ata gtg cag cag cag aac aat ttg ctg agg
gct att gag gcg caa cag cat ctg ttg caa ctc aca gtc tgg ggc atc aag cag ctc cag gca aga atc ctg
tct ggt ggc cgt ggc ggt tgg atg gag tgg gac aga gaa att aac aat tac aca agc tta ata cac tcc tta
att gaa gaa tcg caa aac cag caa gaa aag aat gaa caa gaa tta ttg gaa tta gat aaa tgg cat cac cat
cac cat cac tga
Figure 2.1. Amino acid and DNA sequence of HP construct.
The expression system was BL21(DE3) E. coli cells. The cells were harvested in LB and induced
with 2 mM IPTG for over-expression of the protein of interest. The HP protein has a tendency to
get expressed in the form of inclusion bodies in the cell cytoplasm. Therefore, the purification
was further preceded by inclusion body isolation and solubilization. The cells were collected by
centrifugation of the culture at 9000g. To isolate the inclusion body, the cells were lysed using

38

sonicator. To prevent the protein of interest from degradation, protease inhibitor cocktail
tablet was added in the stock lysis solution. At first the cells were lysed in PBS with 50%
amplitude of sonication, and 0.5 s on and 0.5 s off for 1 min. This cycle was repeated 5 times
with an alternating rest period of 1 min. This was done to avoid any heating of the protein
sample. The inclusion bodies’ comprising the HP protein did not dissolve in PBS, therefore, a
milky white solution was obtained. The inclusion bodies were then collected by centrifugation
at 48000g. This step of PBS solubilization is beneficial to dissolve the unwanted bacterial cell
proteins. Thus, three rounds of PBS solubilization were done by lysis to eliminate most of the
unwanted bacterial protein impurities. After three rounds of sonication in PBS, the cells were
lysed in PBS with 6M GuHCl. The presence of 6M GuHCl solubilized the inclusion bodies
completely. The sonication conditions used were 70 % amplitude with 0.8s on and 0.2s off for 1
min, with 1 min rest period. This cycle was repeated for 5 times. After the sonication, the
solution appeared clear indicating the solubilization of the inclusion bodies in PBS with 6M
GuHCl. The protein solution was then collected by centrifugation at 48000g which eliminated
the cell membrane and debris as a small pellet. All the above mentioned procedure was done at
pH 7.4. The HP protein inclusion bodies could be solubilized in various other conditions and pH
as well; glacial acetic acid at pH 3.0, PBS with 8M Urea at pH 7.4, and PBS with 1% SDS at pH
7.4. The solubility of HP was concluded after observation of clear lysate solution after lysis in
the respective buffers. The solubilized and purified SDS-PAGE gels of HP in glacial acetic acid
(Figure 2.2) and 8M urea are shown below (Figure 2.3). For low pH in glacial acetic acid
purification, RP-HPLC was used and for physiologic pH urea purification, IMAC was used.

39

Figure 2.2. SDS-PAGE gel showing RP-HPLC purified HP in glacial acetic acid at pH 3.0

Figure 2.3. SDS-PAGE gel showing IMAC purified HP in the presence of urea at pH 7.4.
2.4.

Detection of Expression of HM using Solid State NMR of Inclusion Bodies

The gp41 HM plasmid includes the NHR residues 535(M535C)-581, a non-native loop (SGGRGG),
followed by CHR and MPER residues 628-683, and a non-native GGGGHHHH tag. The amino
acid residue numbering is based on the HXB2 Env sequence numbering.

40

CTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILSG
GRGGWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWN
WFNITNWLWYIKGGGGHHHH
atg tgc acg ctg acg gta cag gcc aga caa tta ttg tct ggt ata gtg cag cag cag aac aat ttg ctg agg
gct att gag gcg caa cag cat ctg ttg caa ctc aca gtc tgg ggc atc aag cag ctc cag gca aga atc ctg
tct ggt ggc cgt ggc ggt tgg atg gag tgg gac aga gaa att aac aat tac aca agc tta ata cac tcc tta
att gaa gaa tcg caa aac cag caa gaa aag aat gaa caa gaa tta ttg gaa tta gat aaa tgg gcg tcg ctg
tgg aac tgg ttt aac atc acg aac tgg ctc tgg tac atc aag ggt ggt ggc ggg cat cac cat cac tga
Figure 2.4. Amino acid and DNA sequence of HM.
Initially it was not understood whether the HM protein was expressed or not. To understand
the expression of HM, we implemented solid state NMR on the HM inclusion bodies. The cells
expressing HM were grown in minimal media with addition of 13CO labeled Leu. The Addition of
13

CO-Leu during the expression period resulted in 13CO-labeling of HM. Cells were lysed in PBS

followed by centrifugation of the lysate and the harvested pellet was enriched in any HM IBs.
The 13C NMR spectrum of this pellet showed a prominent 13CO feature consistent with ~300 mg
HM/L in IBs (Figure 2.5). The main bottleneck(s) to purified HM were therefore low
solubilization of HM IBs and/or purification losses.

41

Figure 2.5. Chemical shift corresponding to 13CO- Leu.
Solid state NMR sample preparation and experimental conditions: BL21(DE3) cells expressing
HM protein were grown overnight in 50 mL flask a 37 °C. After overnight growth, the cells were
collected by centrifugation at 9000 g at 4 °C for 15 min. The cells were the resuspended into M9
minimal media also containing 250 µM of 50% v/v glycerol as carbon source, and 100 µL 1.0 M
MgSO4. After one hour of growth, the cells were induced with 2 mM IPTG for over-expression
of HM. Also, 10 mg each of all the essential amino acids were added and 10 mg of 13CO Leu was
added to label the HM at Leu residues. Another addition of the amino acids was done after one
hour. Then the cells were allowed to grow for three hours more, totaling growth time to be as
six hours. The cells were then collected by centrifugation. The cells were lysed with sonication
at 60% amplitude with 0.6s on and 0.4s off. Lysing the cells in PBS at pH 7.4 formed a milky
white solution with inclusion bodies suspension. The inclusion bodies were collected by
centrifugation at 48000 g. The labeled HM inclusion body sample was the frozen in liquid N2
and packed in 4 mm rotor using packing tool.

42

The NMR data were obtained with a 9.4T, triple resonance magic angle spinning (MAS) probe
with a 4 mm rotor. The temperature was maintained at -20 °C with the flow of liquid N2.
Experimental parameters were as follows: (1) 8 kHz MAS frequency, and (2) 5 µs 1H π/2 pulse,
2ms cross-polarization time with 50 kHz 1H field, and 70-80 kHz ramped 13C field. The
spectrum was externally referenced to the methylene carbon of adamantine at 40.5 ppm.
2.5.

Solubilization of HM

The solubilization steps of HM inclusion bodies were same as described in section 2.3 for HP
solubilization. HM is most efficiently soluble in PBS with 6M GuHCl at pH 7.4. Other solubilizing
agents at different pHs did not solubilize HM from inclusion bodies very well; PBS with 8M Urea
at pH 7.4, 10 mM Tris-HCl at pH 9.0, glacial acetic acid at pH 3.0, PBS containing 1% SDS, 2X
CMC of non-ionic detergents like triton, N-decyl-β-D-maltopyranoside, N-lauroyl sarcosine, and
N-octyl-β-D-1-glucopyranoside (Table 2.1). Presence of SDS made HM fully soluble, however,
the protein aggregated with the attempt of removal of SDS in the later stages of purification
and refolding. The solubility of HM was checked by SDS-PAGE of the lysate in the respective
buffers. The SDS-PAGE gel did not show clear difference between the solubility of various
solubilizing agents (Figure 2.6). However, after sonification in the respective buffers the
following were observed: (1) a clear solution in 1% SDS, (2) partially clear solution in 0.5%
Sarkosyl, (3) cloudy solution in the presence of 0.4% triton and (4) cloudy solution in the
presence of 8M urea. The extent of solubility of the protein construct was not determined by
measuring the absorbance at 280 nm. Based on the visual clarity of the protein solution in
respective buffers, the determination of best buffer for maximum solubilization was done.

43

Figure 2.6. Solubilization of inclusion bodies in various solubilizing agents.

44

Solubilizing agent

Solubility

Purification

PBS

Not Soluble

Tris

Not Soluble

Urea

Soluble

Did not bind to His-select resin

SDS

Very Soluble

Protein precipitated out with
the removal of SDS

N-Decyl-β-D-maltopyranoside

Sparingly

N-lauroyl sarcosine

Negligible

N-Octyl β-D-1-glucopyranoside

Negligible

Triton

Negligible

Glacial acetic acid

Soluble

Did not bind to His-select resin

Low

yield

in

RP-HPLC

purification due to binding of
the protein molecules to the
column. The protein eluted out
with varied retention times at
high acetonitrile concentration.

Table 2.1. Solubilization of inclusion bodies containing HM in various solubilizing agents.

45

2.6.

Purification of Gp41 Ectodomain Protein Constructs

2.6.1. HP
After solubilization as explained in section 2.5, the lysate containing HP in PBS + 6M GuHCl in
the presence of protease inhibitor was allowed to mix in His-select cobalt (Co2+) resin for 4
hours at 4 °C. HP has a C-terminal six residue His-tag for binding to the charged metal ion in the
resin. To avoid non-specific binding to the resin, 1 mM imidazole was added to the buffer. After
the binding of the protein with the Co2+ resin, the protein was isolated from the resin by gravity
purification in a column at room temperature. To remove the impurities i. e. the other bacterial
proteins, a gradient of imidazole concentration was used sequentially. Then finally at high
imidazole concentration, i. e. 250 mM, the HP protein was eluted out. The concentration of
eluted protein was measuring the absorbance of the Trp residues at 280 nm. The yield of HP
was ~50 mg/L of culture.
2.6.2. HM
The purification of HM was done in the same procedure as HP explained in section 2.6.1.
However, it was observed that HM did not bind to the Co2+ resin as efficiently as HP. This led us
to believe that probably the six residue His-tag was not exposed for proper binding with the
charged metal ion in the resin. At first a short two residue Gly tag was added before the Nterminal end of His-tag. However, it was observed that the protein construct still did not bind to
the resin (Figure 2.7). Therefore, we added a four residue Gly-tag before the His-tag, i. e. at the
N-terminal of the His-tag by site directed insertion mutation. With the addition of the Gly-tag,
we observed that the protein HM bound very tightly to the resin and could not be eluted out
easily by 500 mM imidazole containing elution buffer. To avoid this tight binding and the low
46

yield, we deleted two His residues from the six residue His-tag by site directed deletion
mutation. Thus the HM protein sequence has the four residues Gly-tag and six residue His-tag.
At this point, we observed that it was still challenging to solubilize HM in solubilizing agents like
3xCMC Sarkosyl (Figure 2.8). We observed that only 6M GuHCl efficiently solubilized HM and
resulted in efficient purification (Figure 2.9). This Gly-His-tag helped to bind the protein to the
resin and could be eluted out with high yield of protein. The yield HM was ~22 mg/L of culture.

Figure 2.7. SDS-PAGE gel showing low solubilization and negligible binding of HM with
2Gly-4His tag in 8M urea with Co2+ resin.

47

Figure 2.8. SDS-PAGE gel showing inefficient binding of HM with 4Gly-4His tag in 3xCMC
Sarkosyl with Co2+ resin.

Figure 2.9. SDS-PAGE gel showing binding of HM with 2Gly-4His tag in 6M GuHCl with Co2+ resin
and elution with high purity. The bands near 25 kD are dimer bands, confirmed by Western blot
with anti-His antibody.

48

2.7.

Western Blot Analysis

The protein purified was verified by Western blot technique. It is a very sensitive technique as it
can detect presence of specific protein in a heterogeneous mixture of proteins. In this method,
the protein sample is first separated by SDS-PAGE. Then the protein bands are transferred to
nitrocellulose membrane. The membrane with protein bands was allowed to stain with
antibodies which can specifically bind to the protein of interest. In our work, the HP and HM
protein samples has a His-tag at the C-terminus. Therefore, we used anti-His-HRP conjugated
antibody. The HRP conjugate facilitates the observation by chemiluminescence by activating the
chemiluminescent substrate. To observe binding of the HIV-1 broadly neutralizing antibodies
4E10 and 2F5 to the MPER epitopes in HM and partial epitope in HP, the antibodies were
allowed to bind to the protein of interest by Western blot. The antibodies 4E10 and 2F5 were
not conjugated to HRP as mention earlier in this section. Therefore, a secondary antibody Goat
Anti-Hamster IgG (H+L) HRP conjugate was used to bind to the 4E10 and 2F5.
2.8.

Peptide Synthesis of N-terminal Fusion Peptide Region of Gp41 Ectodomain

Previous studies have shown that there is less protein expressed with the FP region included in
FP-HP construct (1) . The FP region is a hydrophobic domain and thus could easily get
aggregated in the solution. Also the FP has affinity to bind to the membrane and so binding of
FP to E.coli membranes during purification process leads to more difficulties in the isolation
process. Therefore, the ectodomain construct of gp41 can be designed by ligating (details of
ligation in section. 2.9) the chemically synthesized FP with the expressed HM. The wild type
sequence of HIV-1 HXB2 strain envelope protein from 512-534 (S534A)-thioester linker (FP23
linker) sequence of FP was synthesized by t-Boc synthesis. Solid phase peptide synthesis known
49

as – tert-Butoxycarbonyl (t-Boc) method can be done based on the principles of the work of
Merrifield (2,3). According to this method there is sequential building of peptide chains with the
peptides being anchored to a solid insoluble resin support. To the resin (solid phase support)
the first amino acid is covalently bonded with a C-terminal linker group and the N-terminus is
protected. This is followed by series of washes to remove the contaminants and deprotect the
N-terminus of the attached amino acid. Then a new amino acid is added with an unprotected Cterminal to get attached to the N-terminal of the previous amino acid and build up the
sequence. This whole cycle is repeated until the required sequence is created (2). The Cterminal was modified consisting of a thioester. The whole process of t-Boc synthesis is shown
in Figure 2.10. The prepared peptide can be cleaved from the resin by breaking the covalent
bond between the resin and the first amino acid using hydrofluoric acid. The synthesized
peptide was sent for cleavage from the resin to Midwest Bio-tech Inc. After the cleavage, it was
purified by RP-HPLC and MW was verified by mass spectrometry.

50

Figure 2.10. Schematic representation of t-Boc solid phase peptide synthesis.

From 1 g of Leu-Pam resin, 750 mg of crude peptide was obtained, and after an initial
purification we expect to obtain 240 mg of pure peptide from this synthesis. There are some
disadvantages of solid phase peptide synthesis, such as, as the chain grows in length, the
residues tend to form secondary structure and fold. This might hinder the free N-terminal of
the attached amino acid where the next residue would be added.
2.9.

Production of Full-Length Gp41 Ectodomain Constructs by Native Chemical Ligation

Native chemical ligation (NCL) is an efficient technique to prepare larger protein constructs (1).
This method allows the synthesis of larger constructs by allowing ligation of synthetic peptides
to an expressed protein (4,5). The peptide could be synthesized with t-Boc solid phase peptide
synthesis (details in the section 2.8). In this synthesis the resin used consists of thioester which
would get covalently bonded to the C-terminal amino acid. The principle (4) on which native

51

chemical ligation works is the synthetic peptide with the thioester at the α-carboxyl group
undergoes nucleophilic attack by the side chain of the N-terminal Cys residue of the amino
terminal of the expressed Hairpin protein. The thioester ligated product initially undergoes
rapid intramolecular reaction due to the favorable five-membered ring geometric arrangement
of the α-amino group of the second peptide (expressed HM). This finally yields a product with a
native peptide bond at the site of ligation. Both the ligated peptides (synthetic peptide +
expressed protein) are in unprotected form and needs no further manipulation. There are
several factors on which the rate of ligation depends: (1) the nature of the thiol leaving group,
and (2) side chain steric conditions (3,5). The alkyl thioesters which are used in solid phase
peptide synthesis are comparatively less reactive. This encourages the use of catalyst in this
reaction and the rate of the reaction depends on the choice of the catalyst also. It has been
shown that 4-mercaptophenyl acetic acid (MPAA) catalyses the ligation reaction to promote the
in situ formation of the more reactive thioester moiety (3,5) (Figure 2.11).

52

Figure 2.11. Mechanism of native chemical ligation. The FP23 that is synthesized by tBoc SPPS is shown having a thiol on the C-terminus. To make the reaction more rapid, a
catalyst MPAA is used. MPAA undergoes thioester exchange, and the Hairpin with the Nterminal Cys can easily form thioester-linked intermediate. Finally the more stable
product, i.e., the peptide bond is formed.
In order to obtain the FP-HM construct, the expressed HM protein was ligated with FP. The FP
was dissolved in the ligation buffer (8M Guanidine chloride, 0.1 M phosphate buffer, and pH-7).
The peptide was reacted with 4-mercaptophenyl acetic acid (MPAA) catalyst to modify the
thioester linked to the C-terminus of the fusion peptide (5,6) and the mixture was incubated for
30 min. Similarly, HM was dissolved in the ligation buffer including Tris(2-carboxyethyl)phosphine (TCEP) (2mM) to maintain cysteine residue in reduced state, and the two solutions
were mixed together. The reaction was allowed to proceed for 2 days in an inert (Ar)
atmosphere and ambient temperature. This was followed by purification by RP-HPLC with a C4
columm (6). Three peaks comprising of FP23, HM, and FP-HM were collected and MW was
verified by mass spectrometry. Acetonitrile was removed from the eluted FP-HM fraction and
allowed to concentrate.

53

2.10. Stabilization of Gp41 Ectodomain Protein Constructs
The refolding and stabilization of the purified protein were done by dialyzing against 10 mM or
50 mM sodium formate buffer at pH 3.2 containing 150 mM NaCl + 0.2 mM TCEP reducing
agent at 4°C. The HP and HM protein were diluted to ~0.1 mg/mL concentration and then
subjected to dialysis for removal of the 6M GuHCl solubilizing agent. After at least three buffer
exchanges, the protein sample was concentrated to ~1 mg/mL. The solubility of HP and HM at
low pH 3.2 without any solubilizing agent was verified by centrifugation and then measuring the
absorbance of the supernatant at 280 nm. The secondary structure of the refolded protein
constructs were verified by measuring the mean residue molar ellipticity by circular dichroism
(CD). HP and HM protein constructs were also dialyzed against phosphate buffers ranging from
pH 5-7.4 and Tris-HCl buffer at pH 9.0. However, the proteins aggregated indicating no
solubility. Aggregation of the protein constructs was concluded by centrifuging the sample in
respective buffer at 16000g for 5 min and measuring the protein concentration in the
supernatant. One of our aims was to attain the gp41 ectodomain constructs HP and HM in
physiological pH. The solubility of HP and HM was also tested in the presence of detergent like
n-dodecylphosphocholine (DPC). It was observed that even in the presence of DPC, HP and HM
were soluble at low pH ~4.0 and not at pH 7.4. We therefore studied the effect of solubilizing
agent to solubilize at physiological pH. For this purpose 6M GuHCl was used. In the presence of
6M GuHCl, both the HP and HM were soluble at physiological pH 7.4 as well as low pH 3.2. The
global secondary structure of the HP and HM showed folded structure i. e. α-helical. The αhelical curve has expected minima at 222 nm and the minimum at 208 nm is distorted due to
the GuHCl effect.

54

2.11. Determination of Secondary Structure of Gp41 Ectodomain Constructs
2.11.1. Circular Dichroism
CD is a very useful spectroscopic tool to verify global secondary structure of large biological
molecules. In this technique the difference in absorption of left and right circularly polarized
light by chiral molecules is recorded and plotted against the wavelength. Percent helicity can be
calculated from the mean residue molar ellipticity (θ222) at wavelength 222 nm (minima). θ222 of
-33,000 deg cm2 dmol-1 at wavelength 222 nm is considered to be 100% (7). The thermal
stability of the protein constructs can also be measured by observing the CD signal at 222 nm as
a function of temperature with 2 °C steps between 10 °C – 100 °C.

2.11.2. Solid State Nuclear Magnetic Resonance
X-ray crystallography and solution state NMR are powerful techniques which generally give high
resolution structures. There are many x-ray crystallography (8-12) and liquid state NMR (13)
structures of different parts of the gp41. However, there are no structures of the full length
gp41 in physiologically relevant membrane environment. X-ray crystallography and solution
state NMR generally do not allow the study of membrane proteins in membrane environment.
Gp41 is a membrane protein and it would be ideal to study the structure in natural membrane
environment. Solid state NMR is an ideal technique that can be used for studying membrane
proteins structure in membrane mimetic environment. Our group has been successful in

55

determining structures of the fusion peptide of the gp41 in membranes by solid state NMR (1417).
Analysis of the chemical shifts of the α-carbon, β-carbon, or the carbonyl carbon (CO) for a
specific amino acid residue can reveal the structural information for that particular residue in
the context of the protein. The chemical shift of each of these carbons is correlated with the
likely residue conformation to get the information whether the residue is in the helical, and βstrand conformation (18).
Rotational echo double resonance (19) (REDOR) is a high-resolution solid state NMR experiment
which can be used for observing the chemical shift of a specific carbonyl carbon in the entire
protein molecule. In order to observe a specific site in the protein, a unique sequential pair
should be chosen. In our case, two schemes of labeling will be performed. (i) Labeling of
synthesized FP: For example, to have

13

CO labeled leucine and

15

NH labeled phenylalanine in

the FP, accordingly labeled amino acids can be used during peptide synthesis. (ii) Labeling of
expressed HP-MPER: For instance,
15

13

C labeling all of the carbonyl carbons of aspartic acid and

N labeling all of the amide nitrogens of lysine is possible. Whenever there is an Asp-Lys in the

protein sequence, there will be two NMR active nuclei connected to each other. A particular
REDOR pulse sequence (Figure 2.12) would allow us to have acquisition of signal (S0) from all
the 13C in the protein sample. At half rotor period a 15N π-pulse is given and a dipolar dephased
signal (S1) is produced. The difference signal (S0-S1) is due to 13C – 15N dipolar coupling through
space. Thus selective labeling process coupled with REDOR pulse program provides a powerful
tool for predicting the secondary structure of a particular amino acid in the entire protein. This

56

is attained by comparing the chemical shift obtained from the protein sample and the
databases of the 13CO chemical shifts (18).

Figure 2.12. The REDOR pulse sequence.
2.12. Determination of Oligomeric State(s) by Size Exclusion Chromatography
The oligomeric property of protein molecules can be determined by Size Exclusion
Chromatography (SEC). The chromatographic method SEC is based on separation of particles
based on the size or the molecular weight. The sample is allowed to pass through a matrix
typically made up of polysaccharide cross-linking forming pores of various sizes. The matrix is
the stationary phase and the sample to be characterized is dissolved in the mobile phase. The
criterion for the matrix material is that it should not interact with the sample in the mobile
phase. The rate of flow of the sample depends on the size of the particles. The protein
molecules of larger size elute earlier and the smaller molecules elutes later. The elution curve in
the chromatogram resembles Gaussian distribution. The calibration of the matrix columns were
done by using protein samples of known molecular weight as mobile phase (Figure 2.13).
The calculations for calibration are as follows:
Partition coefficient Kav = (Ve – Vo)/(Vc – Vo)

where Ve = elution volume
57

Vc = geometric column volume
Vo = column void volume
The linear calibration curve is plotted as Kav as a function of Log10(MW).

Figure 2.13. SEC A280 of MW standards loaded on (A) Superdex 75 and (B) Superdex 200 columns. The
protein standards used were (A) Conalbumin (75 kDa), Ovalbumin (44 kDa), Carbonic Anhydrase (29
kDa), Ribonuclease A (13.7 kDa), and Aprotinin (6.5 kDa) and (B) Thyroglobulin (669 kDa), Ferritin (440
kDa), Aldolase (158 kDa), Conalbumin (75 kDa), Ovalbumin (44 kDa), Carbonic Anhydrase (29 kDa),
Ribonuclease A (13.7 kDa), and Aprotinin (6.5 kDa)The Vo is the void volume and corresponds to 2 MDa
Blue Dextran. Kav vs log10(MW) for the (C) Superdex 75 and (D) Superdex 200 columns. The Kav = (Ve –
Vo)/(Vc – Vo) where Ve and Vc are the elution and column volumes, respectively.
58

2.13. Antibody binding studies by Direct Immunoprecipitation
We had used the technique immunoprecipitation to detect the binding of specific broadly
neutralizing antibodies to epitopes in the proteins HP and HM. Immunoprecipitation is a
technique which can separate a protein antigen-antibody complex from a solution. The antigenantibody complex is immobilized on a solid phase and later collected. We had implemented the
technique of Direct Immunoprecipitation to ensure that the protein antigen binds specifically to
the immobilized antibodies. In direct immunoprecipitation the antibodies are first allowed to
bind to the protein A/G beads. The beads used were magnetic beads with covalently linked
protein G on them. Protein G covalently binds to the antibody heavy chain. The protein of
interest is then allowed to bind to the immobilized antibody. This also verifies whether the
epitope for the antibodies is present in the protein of interest or not. We have used 4E10 and
2F5, the broadly neutralizing antibodies among various strains of HIV to bind to HP and HM
constructs of HIV-1. The experimental steps and conditions are as follows:
Binding of Antibodies – The protein G magnetic beads were washed with PBS + 1% Tween20 at
pH 7.4 and then antibody was added to bind to the beads. It was observed that even though
excess of antibody was added, there was still the presence of some unbound beads which could
bind to the protein molecules leading to misinterpretation of results. We therefore, allowed IgG
to bind to the protein G beads after the binding of 4E10 or 2F5. With this step, the unbound
beads were covered with IgG. IgG binds to protein G very efficiently. The control experiments
were done with only IgG.
Wash – The beads were then washed to remove the unbound antibodies.
59

Binding of Protein – The protein samples HP or HM were added to allow binding to the
antibodies by gently mixing for 2 hours. The whole sample volume of 700 μL was maintained at
pH 7.4.
Extraction of Protein – The protein was extracted from bound antibody-protein complex
immobilized on beads by collecting the beads and boiling in SDS buffer for ~5 min. The
extracted protein sample was then run on 15% SDS-PAGE gel to verify the presence of our
protein of interest.
2.14. Vesicle fusion Assays using Fluorescence Spectroscopy
POPC: POPG:Chol 8:2:5 mole was dissolved in chloroform followed by chloroform removal
with nitrogen gas and vacuum pumping. The film was homogenized by freeze-thaw cycles in ~1
mL buffer and extruded through 100 nm diameter pores to form unilamellar vesicles.
Fluorescently labeled vesicles were similarly prepared except that the mixture also contained 2
mole% of the fluorescent lipid N-NBD-PE and 2 mole% of the quenching lipid N-Rh-PE. Labeled
and unlabeled vesicles were mixed in 1:9 ratio with total [lipid + Chol]  230 μM. Experiments
were also done using positively-charged vesicles with POPC:DOTAP:Chol in same mole ratio.
Fluorescence of the stirring vesicle solution was measured at 37 oC with 467 nm excitation, 530
nm detection, and 1 s time increment. After measurement of the baseline fluorescence F0, a
protein aliquot was added and marked time t = 0. Vesicle fusion was reflected in the increased
fluorescence ΔF(t) = F(t) – F0 due to longer distances between fluorescent and quenching lipids
in a fused (labeled+unlabeled) vesicle relative to the initial labeled vesicle. The dead-time in the
assay was ~5 s and asymptotic fluorescence (ΔFf ) was reached after ~600 s. The maximum
60

fluorescence change (ΔFmax) was detected after addition of 12 μL 10% Triton X-100 which
solubilized the vesicles. Percent fusion was M(t) = [F(t)/F(max)]x100. Comparison among
assay replicates showed (Mf )/Mf  0.02.
We wanted to compare vesicle fusion induced by HP, HM, and FP-HM where all proteins were
in the same stock buffer conditions. FP-HM was not soluble without 6 M GuHCl, so the chosen
stock conditions were 10 mM sodium formate at pH 3.2, 6 M GuHCl, and 0.2 mM TCEP. The
effects of the stock pH and GuHCl were minimized by always adding 7.5 μL of stock into a final
total volume of 1200 μL. Therefore the final conditions were maintained as physiological, e.g.
pH 7.4 with very low [GuHCl] = 0.04 M. Vesicle fusion was performed for final pHs of 3.2 and 7.4
with respective stock [protein] = 20 μM and 160 μM, chosen so that (1) for all proteins, Mf
<100% with no light scattering; and (2) for at least one protein, Mf was appreciably greater than
0%.

61

REFERENCES

62

REFERENCES
1.

Sackett, K., Wexler-Cohen, Y., and Shai, Y. (2006) Characterization of the HIVN-terminal
fusion peptide-containing region in context of key gp41 fusion conformations. Journal of
Biological Chemistry 281, 21755-21762

2.

Merrifield, R. B. (1963) Solid Phase Peptide Synthesis .1. Synthesis of a Tetrapeptide. J
Am Chem Soc 85, 2149-&

3.

Hackeng, T. M., Griffin, J. H., and Dawson, P. E. (1999) Protein synthesis by native
chemical ligation: Expanded scope by using straightforward methodology. Proc Natl
Acad Sci USA 96, 10068-10073

4.

Dawson, P. E., Muir, T. W., Clarklewis, I., and Kent, S. B. H. (1994) Synthesis of proteins
by native chemical ligation. Science 266, 776-779

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Johnson, E. C. B., and Kent, S. B. H. (2006) Insights into the mechanism and catalysis of
the native chemical ligation reaction. J Am Chem Soc 128, 6640-6646

6.

Sackett, K., Nethercott, M. J., Shai, Y., and Weliky, D. P. (2009) Hairpin Folding of HIV
gp41 Abrogates Lipid Mixing Function at Physiologic pH and Inhibits Lipid Mixing by
Exposed gp41 Constructs. Biochemistry 48, 2714–2722

7.

Greenfield, N. and Fasman, G. D. (1969) Computed Circular Dichroism Spectra for
Evaluation of Protein Conformation. Biochemistry 8, 4108-&

8.

Liu, J., Deng, Y. Q., Dey, A. K., Moore, J. P., and Lu, M. (2009) Structure of the HIV-1 gp41
Membrane-Proximal Ectodomain Region in a Putative Prefusion Conformation.
Biochemistry 48, 2915-2923

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Chan, D. C., Fass, D., Berger, J. M., and Kim, P. S. (1997) Core structure of gp41 from the
HIV envelope glycoprotein. Cell 89, 263-273

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Weissenhorn, W., Dessen, A., Harrison, S. C., Skehel, J. J., and Wiley, D. C. (1997) Atomic
structure of the ectodomain from HIV-1 gp41. Nature 387, 426-430

11.

Shi, W., Bohon, J., Han, D. P., Habte, H., Qin, Y., Cho, M. W., and Chance, M. R. (2010)
Structural Characterization of HIV gp41 with the Membrane-proximal External Region.
Journal of Biological Chemistry 285, 24290–24298

12.

Buzon, V., Natrajan, G., Schibli, D., Campelo, F., Kozlov, M. M., and Weissenhorn, W.
(2010) Crystal Structure of HIV-1 gp41 Including Both Fusion Peptide and Membrane
Proximal External Regions. Plos Pathogens 6

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Song, L., Sun, Z.-Y. J., Coleman, K. E., Zwick, M. B., Gach, J. S., Wang, J.-h., Reinherz, E. L.,
Wagner, G., and Kim, M. (2009) Broadly neutralizing anti-HIV-1 antibodies disrupt a
hinge-related function of gp41 at the membrane interface. PNAS 106, 9057–9062

14.

Sackett, K., Nethercott, M. J., Epand, R. F., Epand, R. M., Kindra, D. R., Shai, Y., and
Weliky, D. P. (2010) Comparative Analysis of Membrane-Associated Fusion Peptide
Secondary Structure and Lipid Mixing Function of HIV gp41 Constructs that Model the
Early Pre-Hairpin Intermediate and Final Hairpin Conformations. Journal of Molecular
Biology 397, 301–315

15.

Bodner, M. L., Gabrys, C. M., Parkanzky, P. D., Yang, J., Duskin, C. A., and Weliky, D. P.
(2004) Temperature dependence and resonance assignment of C-13 NMR spectra of
selectively and uniformly labeled fusion peptides associated with membranes. Magn
Reson Chem 42, 187-194

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Yang, J., and Weliky, D. P. (2003) Solid-state nuclear magnetic resonance evidence for
parallel and antiparallel strand arrangements in the membrane-associated HIV-1 fusion
peptide. Biochemistry 42, 11879-11890

17.

Yang, J., Parkanzky, P. D., Bodner, M. L., Duskin, C. A., and Weliky, D. P. (2002)
Application of REDOR subtraction for filtered MAS observation of labeled backbone
carbons of membrane-bound fusion peptides. J Magn Reson 159, 101-110

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Zhang, H. Y., Neal, S., and Wishart, D. S. (2003) RefDB: A database of uniformly
referenced protein chemical shifts. J Biomol Nmr 25, 173-195

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Gullion, T. (1998) Introduction to rotational-echo, double-resonance NMR. Concept
Magnetic Res 10, 277-289

64

CHAPTER 3
Structural and Functional Characterization of Gp41 Ectodomain

65

3.1

Introduction:

HIV is enveloped by a membrane obtained during viral budding from an infected host cell.
An early step in infection of another cell is fusion of the viral and cell membranes with
accompanying release of the viral nucleocapsid into the cytoplasm (1). The HIV membrane
includes a gp160 glycoprotein complex comprised of two noncovalently associated
subunits, gp120 and gp41 (2). Gp41 is a ~350-residue monotopic integral membrane protein
with a ~180-residue ectodomain (Figure 3.1). Gp120 is bound to the gp41 ectodomain. HIV
targets lymphocytes via binding of gp120 to cell receptors and gp120 moves away from or
dissociates from gp41. Gp41 then undergoes large conformational changes with
accompanying catalysis of membrane fusion. To our knowledge, the gp120/cell receptor
complex is only for target cell identification and gp41 is the only fusion protein. Much of our
biophysical understanding of fusion has therefore come from studies of gp41 with an
emphasis on its ectodomain which can contact the outer leaflets of both the viral and cell
membranes. Mutagenesis has demonstrated that there are two ~20-residue regions of the
gp41 ectodomain that play key roles in fusion likely through membrane interaction (3,4).
The N-terminal fusion peptide (FP) and C-terminal membrane-proximal external region
(MPER) are postulated to bind to the host cell and viral membranes, respectively. The fusion
significances of the FP and MPER have been supported by observation of vesicle fusion
induced by FP or MPER peptides (5,6). There is an intervening N-heptad repeat (NHR), loop,
and C-heptad repeat (CHR) between the FP and MPER domains (Figure 3.1).

66

Electron micrographs of virions show clusters of three gp160, i.e. three gp120 and three gp41
molecules. These clusters are likely the initial protein state before any changes in membrane
topology due to membrane fusion (7). The extraviral region of gp160 is termed gp140 and is
comprised of gp120 and the gp41 ectodomain without the gp41 TM and gp41 endodomain. WT
Gp140 is typically monomeric but gp140 trimers can be stabilized via mutations and a
gp120/gp41 ectodomain cross-link (8,9). There are ~5 Å structures of such gp140 trimers that
likely represent the protein state prior to membrane fusion (10-12). The structure includes a
loose bundle of three parallel NHR helices and three CHR helices forming a tripod. The
monomer structure is NHR-helix/70o-turn/CHR-helix. The FP and MPER are not in the structure.
There are also atomic-resolution structures of segments of the gp41 ectodomain typically
without the FP and MPER and without gp120 (13-16). These show NHR-helix/180o-turn/CHRhelix hairpin structure as well as assembly of three molecules into a six-helix bundle (SHB) with
the three NHRs forming parallel coiled-coil structure on the bundle interior and the three CHRs
packing antiparallel to the NHRs on the bundle exterior. Melting temperatures of ~70 o C are
observed for shorter ectodomain constructs with hairpin structure whereas temperatures up to
110o C are observed for longer constructs (17,18). This thermostability has supported the SHB
as the final gp41 structure during fusion.
In addition to the initial NHR/70o-turn/CHR bent structure and the final NHR/180o-turn/CHR
SHB structure, a “pre-hairpin intermediate” (PHI) structure has been proposed to form after
removal of the gp120s. Each PHI gp41 has a fully extended (no turn) structure, i.e. NHRhelix/0o-turn/CHR-helix and there are separate NHR and CHR trimer helical bundles. To our
knowledge, the existence of the gp41 PHI is only supported by functional studies, in particular
67

inhibition of membrane fusion and HIV infection with NHR or CHR+MPER peptides (19,20).
These peptides are proposed to bind to the CHR and NHR bundles of the PHI, respectively, and
to inhibit the PHISHB structural transition. CHR+MPER peptides are a clinically-prescribed HIV
treatment (21).
There are also distinct membrane structures during fusion (22). The separate viral and host cell
membranes first merge into a hemifusion intermediate characterized by intermembrane lipid
mixing and no contents mixing. This is followed by breaking the hemifusion barrier and
formation of a small pore through which small species (e.g. atomic ions) can pass. The fusion
pore then expands to create a single membrane enclosing the cell and the viral capsid.
There are little data about the relative timing of gp41 and membrane structural changes. One
common model I has been: (1) gp120 receptor binding followed by gp120 removal; (2)
formation of extended PHI gp41 trimer followed by FP insertion into the host cell membrane;
(3) PHISHB trimer folding that brings the two membranes close together; (4) hemifusion; (5)
initial pore formation; and (6) fusion pore expansion (23). The appealing intuitive aspect of this
model is that some of the free energy released during PHISHB folding is used to form
membrane intermediates. However, the relative timings of this model are not supported by the
observation that CHR+MPER peptides inhibit fusion up to the final fusion pore expansion step
(20). Because the peptides are presumed to bind to the PHI trimer but not the SHB trimer,
these data suggest an alternative model II: (1) gp120 receptor binding followed by gp120
removal; (2) formation of extended PHI gp41 trimer followed by FP insertion into the host cell

68

membrane; (3) hemifusion; (4) initial pore formation; (5) PHISHB trimer folding; and (6)
fusion pore expansion (24).
In the present work, we show that the gp41 ectodomain can form stable hairpin monomers as
well as stable hexamers that are likely composed of two SHB trimers. CHR+MPER inhibitor
peptides likely bind to the monomer but not the trimer or hexamer. These findings are the basis
of a new model III (Figure 3.19): (1) gp120 receptor binding followed by gp120 removal; (2)
dissociation of gp41 ectodomain into monomers and formation of extended PHI gp41
ectodomain monomer followed by FP insertion into the host cell membrane; (3) PHIhairpin
monomer folding that brings the two membranes close together; (4) hemifusion; (5) initial pore
formation; (6) hairpin monomerSHB trimerhexamer ectodomain assembly; and (7) fusion
pore expansion. Like model I and unlike model II, the new model III retains the appealing
coupling of the PHISHB transition to initial steps of membrane fusion. There are more
reasonable coordinated changes of the ectodomain and membrane topologies for PHISHB
monomer folding of model III than for PHISHB trimer folding of models I and II. Finally, the
discovery of stable hexamers correlates with other data supporting a requirement of multiple
gp160 trimers for membrane fusion and HIV infection (25,26).
Much of our understanding of the FP and MPER regions of the gp41 ectodomain has been
based on studies of vesicle fusion induced by peptides (27-29). Rapid (~5 s) fusion typically
requires 500-1000 peptides per ~100 nm diameter vesicle which is much higher than the ~30
gp160 per virion and suggests that there are aspects of viral fusion unaccounted for in the
peptide studies (30). A reasonable hypothesis with some supporting data is that the rest of the

69

ectodomain plays an important role in fusion. In the present work, we demonstrate efficient
vesicle fusion with only ~15 gp41 per vesicle provided that the FP, hairpin, and MPER are
included in the large gp41 ectodomain construct. To our knowledge, this is the first
demonstration of fusion synergy between the FP, hairpin, MPER in a large gp41 ectodomain
construct. Such synergy correlates with postulated binding of FP and MPER regions to host cell
and viral membranes, respectively, and also with postulated FP/MPER interaction in a folded
hairpin structure.
The MPER is the epitope of several broadly-neutralizing antibodies (bNAbs) that prevent
infection by diverse isolates of HIV (4,31). There has consequently been continued effort to
develop a HIV vaccine with a MPER immunogen. The ectodomain with MPER and hairpin
structure is a candidate immunogen in part because of the stability of this structure. However,
there is disagreement in the literature about the antigenicity of the hairpin, i.e. whether bNAbs
bind well to the MPER in this structure (32-34). The present study shows such binding for the
hairpin ectodomain which is initially in a monomer or hexamer state.
3.2

Experimental Procedures

3.2.1. HP and HM inserts
Amino acid (Figure 3.1B) sequences are for the HXB2 laboratory strain of HIV and are described
using gp160 numbering. HP and HM include the NHR residues 535(M535C)-581 and a nonnative loop (SGGRGG). HP includes CHR residues 628-666 and a non-native H6 tag. HM includes
CHR+MPER residues 628-683, a non-native G4 spacer, and a non-native H4 tag. The HP insert in
the pGEM-t vector without the H6 tag has been previously described and the H6 tag was then
70

added via PCR (35). The HM insert was generated from the HP insert via multiple rounds of PCR.
The HM DNA sequence is shown in figure 3.2.

Figure 3.1. (A) Schematic diagram of HIV gp41 where FP  fusion peptide, NHR  N-heptad
repeat; CHR  C-heptad repeat, MPER  membrane-proximal external region, TM 
transmembrane domain, and Endo  endodomain. (B) Amino acid sequences of HP, HM, and
FP-HM. The epitopes of the 2F5 and 4E10 bNAbs are marked.

Figure 3.2. DNA sequence of the HM insert

71

3.2.2. HP and HM Expression, Solubilization, and Purification
The expression host was E. coli, BL21(DE3) strain. The typical protocol began with addition of 1
mL of bacterial glycerol stock to 50 mL of LB medium. After overnight growth at 37 oC, the 50
mL culture was added to 1 L of fresh LB medium. Growth was continued for two hours with a
final OD600 of ~0.8. Protein expression was induced with addition of 2 mM IPTG and continued
for 6 hours at 37 oC. “Pellet I” (~9 g) was harvested by centrifugation at 9000g. The next steps
were done at 4 oC using 30 mL PBS at pH 7.4 with protease inhibitor cocktail. 3 g of pellet I was
suspended in PBS and lysed by tip sonication in an ice bath. The lysate was centrifuged at
48000g and the resultant “pellet II” was sonicated in PBS with subsequent centrifugation. SDSPAGE showed that the resultant “pellet III” had a high mass fraction of recombinant protein
(RP). Pellet III was effectively solubilized by sonication in PBS + 6M GuHCl and the RP in this
solution was purified at ambient temperature by affinity chromatography with Co2+ resin. The
resin suspension solutions were PBS (pH 7.4) + 6M GuHCl + imidazole and resin was isolated
using gravity filtration. After initial protein binding with 1 mM imidazole, weakly-bound
proteins were removed using sequential washes with 5 mM imidazole (2), 10 mM imidazole
(2), and 20 mM imidazole (2). The RP was eluted using 250 mM imidazole (4) and the
purified yields of HP and HM were ~50 and ~15 mg/L culture, respectively, as determined by
A280. Elutions were: (1) diluted to ~0.1 mg RP/mL in PBS + 6 M GuHCl + 2 mM DTT; (2) dialyzed
at 4 oC against 50 mM sodium formate buffer (pH 3.2) + 150 mM NaCl + 0.2 mM TCEP reducing
agent; and (3) concentrated to ~1 mg/mL. HP and HM were aggregated in the pH 5 – 9 range in
the absence of GuHCl. The newly constructed HM MW was also confirmed by MALDI (Figure

72

3.3A). The theoretical MW of HM is 13738 Da and according to MALDI it was obtained as 13728
Da. The error of the observed mass from the theoretical mass is 0.073%.
3.2.3. Synthesis and Purification of FP-HM
FP23 (residues 512-534 (S534A)-(thioester linker)) was synthesized manually by t-boc
chemistry, purified by RP-HPLC, and quantitated with the BCA assay Purity was >95% by MALDI
MS. Native chemical ligation between FP23 and HM was done with 1.2 mM FP23, 1.2 mM HM,
0.1 M sodium phosphate buffer at pH 7.2, 8 M GuHCl, 60 mM MPAA, 2 mM TCEP, 1 day time,
and ambient temperature. The ligation products were purified by RP-HPLC. The MW of the
ligated product FP-HM was verified by SDS-PAGE and MALDI experiments (Figure 3.3B and inset
and 3.4A). The theoretical value of MW of FP-HM is 15829 Da and observed value was 15815
Da. The error of the observed mass from the theoretical mass is 0.088%. The difference in mass
of HM and FP-HM from the theoretical value is probably due to the calibration error. The
second round of purification by RP-HPLC shows only FP-HM peak (Figure 3.4B). However, the
yield was very low, therefore, worked with the 1st round purified product containing ~40% of
HM mixed in FP-HM.

73

Figure 3.3. MALDI mass spectra of (A) the HM and (B) FP-HM peaks from the first HPLC
purification. Inset: SDS-PAGE where the middle lane is the FP-HM peak of figure 3.3B and the
right lane is purified HM.

Figure 3.4. (A) First-round RP-HPLC profile of FP+HM ligation. (B) Second-round RP-HPLC of FPHM peak from panel A.
74

3.2.4. Size-Exclusion Chromatography (SEC)
A DuoFlow Pathfinder 20 instrument (Bio-Rad) was used with a flow rate of 0.3 mL/min, A280
detection, and Tricorn semi-preparative columns (GE Technologies).
3.2.5. CD Spectroscopy
A Chirascan spectrometer (Applied Photophysics) was used with a quartz cuvette with 1 mm
pathlength. There were 0.5 nm steps with 1.5 s per step. For each sample, three scans were
averaged. The final spectrum was the (RP + buffer) – buffer difference spectrum.
3.2.6. Western Blots.
Purified RP in SDS buffer (~0.5 mg/mL) was boiled followed by SDS-PAGE (~5 μg RP per lane)
and transfer to a nitrocellulose membrane and incubation in a 10 mL solution containing
antibody (5 μg), TBST at pH 7.4, and 5% w/v nonfat dry milk. Subsequent incubation with Goat
Anti-Hamster IgG (H+L) HRP conjugate secondary antibody (1 μg) was followed by development
with SuperSignal West Pico chemiluminescent substrate.
3.2.7. Immunoprecipitation
5 μg quantities of RP and antibodies were used from 1 mg/mL stocks. Solutions other than RP
stock contained PBS at pH 7.4. bNAb was incubated with Protein G magnetic beads followed by
3 wash removal of free bNAb. Because RP binds to unpassivated beads but not IgG, the beads
were incubated with IgG and then washed. The beads were then incubated with RP in 700 μL

75

solution for 1 hour and washed. Bound RP and antibodies were removed by boiling in SDS
sample buffer and analyzed by SDS-PAGE.
3.2.8. Protein-Induced Vesicle Fusion
Lipid:Chol (1.0:0.5 mole) was dissolved in chloroform followed by chloroform removal with
nitrogen gas and vacuum pumping. The film was homogenized by freeze-thaw cycles in ~1 mL
buffer and extruded through 100 nm diameter pores to form unilamellar vesicles. Fluorescently
labeled vesicles were similarly prepared except that the mixture also contained 2 mole% of the
fluorescent lipid N-NBD-PE and 2 mole% of the quenching lipid N-Rh-PE. Labeled and unlabeled
vesicles were mixed in 1:9 ratio with total [lipid+Chol]  230 μM. Fluorescence of the stirring
vesicle solution was measured at 37 oC with 467 nm excitation, 530 nm detection, and 1 s time
increment. After measurement of the baseline fluorescence F0, a protein aliquot was added and
marked time t = 0. Vesicle fusion was reflected in the increased fluorescence ΔF(t) = F(t) – F0 due
to longer distances between fluorescent and quenching lipids in a fused (labeled+unlabeled)
vesicle relative to the initial labeled vesicle. The dead-time in the assay was ~5 s and asymptotic
fluorescence (ΔFf ) was usually reached by ~600 s. The maximum fluorescence change (ΔFmax)
was detected after addition of 12 μL 10% Triton X-100 which solubilized the vesicles. Percent
fusion was M(t)={ΔF(t)/ΔFmax}×100. Comparison among assay replicates showed (Mf )/Mf 
0.02.
We wanted to compare vesicle fusion induced by HP, HM, and FP-HM where all proteins were
in the same stock buffer conditions. FP-HM was not soluble without 6 M GuHCl, so the chosen
stock conditions were 10 mM sodium formate at pH 3.2, 6 M GuHCl, and 0.2 mM TCEP. The
76

effects of the stock pH and GuHCl were minimized by always adding 7.5 L of stock into a final
total volume of 1200 L with final [GuHCl] = 40 mM. Vesicle fusion was done for final pHs of
3.2 and 7.4 with respective stock [protein] = 20 M and 160 M, chosen so that (1) for all
proteins, Mf <100% with no light scattering; and (2) for at least one protein, Mf was appreciably
greater than 0%.
3.3.

Results

3.3.1. High-yield Protein Production
Cells that had expressed HP were lysed in PBS but SDS-PAGE of the soluble lysate did not show
a clear HP band. It was therefore concluded that most of the HP was in inclusion bodies (IBs).
After an additional lysis in PBS, different solubilization conditions were tested for “pellet III” as
defined in the Experimental section. Similar intensity HP bands were observed in SDS-PAGE of
the lysates from glacial acetic acid, 1% w/v SDS, 8 M urea, or 6 M GuHCl. Purification of the 6 M
GuHCl lysate yielded HP with high purity (Figure 3.2B). The most intense band was the HP
monomer and there were also reproducible weaker dimer bands confirmed to be HP by
Western blotting with anti-His tag antibody. The purified yield was ~50 mg HP/L culture.
The first HM construct had a H6 C-terminal tag without glycines and the initial efforts to
solubilize and purify the protein were unsuccessful as judged by no clear band in SDS-PAGE. It
was unclear whether the main problem was low expression, low solubilization, or purification
losses. HM expression prior to solubilization was then quantitated with a recently-developed
solid-state NMR (SSNMR) method (36). Addition of

13

CO-Leu during the expression period

resulted in 13CO-labeling of HM. Cells were lysed in PBS followed by centrifugation of the lysate
77

and the harvested pellet was enriched in any HM IBs. The

13

C NMR spectrum of this pellet

showed a prominent 13CO feature consistent with ~300 mg HM/L in IBs (Figure 3.5A). The main
bottleneck(s) to purified HM were therefore low solubilization of HM IBs and/or purification
losses.

78

Figure 3.5. (A) 13C SSNMR spectra of a cell pellet harvested from centrifugation of a cell lysis in
PBS. The E. coli cells contained a plasmid with the HM insert and expression was induced for
two hours in minimal medium containing 13CO-Leu. Any expressed HM will therefore be 13COLeu labeled. The SSNMR acquisition parameters included a 9.4 T magnetic field, 4 mm diameter
rotor, 8 kHz magic angle spinning frequency, and ~1 day of signal averaging. The ratio of
integrated isotropic 13CO intensity (peaked at ~175 ppm) to integrated aliphatic intensity (0 –
90 ppm region) translates to expression of ~300 mg HM/L culture. (B) SDS-PAGE of purified HM
(MW = 13.7 kDa) and HP (MW = 11.6 kDa). (C) MALDI MS of FP-HM ligation product twicepurified by RP-HPLC. The experimental ratio [m/z(FP-HM):m/z(HM)] is 1.1522 and matches the
ratio of expected MWs, [MW(FP-HM):MW(HM)] = 1.1522.
A systematic search was carried out to find conditions for solubilization of HM IBs. Two
sequential lyses were done in PBS to solubilize other proteins. Assessment of solubilization of
79

HM IBs in pellet III was done by: (1) visual reduction in pellet size; and (2) SDS-PAGE of the
solution. Many conditions that solubilized HP IBs did not solubilize HM IBs. Appreciable HM
solubilization was only achieved with 1% SDS or 6 M GuHCl and the latter additive was chosen
for solublization and purification. HM with H6 tag did not bind the Co2+ resin whereas HM with a
G4H6 tag bound so tightly that there was no elution with 250 mM imidazole. HM with a G 4H4 tag
both bound tightly to the resin with 20 mM imidazole and also eluted from the resin with 250
mM imidazole. The purified yield was ~15 mg HM/L culture and SDS-PAGE showed dominant
monomer and weaker dimer bands (Figure 3.5B).
FP-HM was produced by native chemical ligation between FP and HM and purified by RP-HPLC.
For one round of purification, MS intensities showed FP-HM:HM1.2 which correlated with
SDS-PAGE. For two rounds of RP-HPLC, the FP-HM:HM10 (Figure 3.5C). However, there was
too little FP-HM, so the FP-HM+HM mixture after only one round was used for subsequent
experiments.
3.3.2. Solubility Only at pH 3 or With 6 M GuHCl.
Solubility in a particular buffer was examined by: (1) dialyzing the protein (~0.2 mg/mL) against
the buffer for one day; (2) centrifugation at 16000g for 5 minutes; and (3) measuring protein
concentration in the supernatant. A protein was typically either soluble by the criterion [final
concentration]/[initial concentration] > 0.8 or poorly soluble with obvious precipitation.
None of the proteins were soluble in: (1) 10 mM sodium formate at pH 5.0; (2) 5 mM HEPES/10
mM MES at pH 7.4; (3) PBS at pH 7.4 with or without 0.1% (w/v) non-ionic detergent (Triton X-

80

100, N-lauroylsarcosine, n-decyl--D-maltopyranoside, and n-dodecylphosphocholine were
tested); or (4) 10 mM Tris at pH 9.0. Both HP and HM were soluble in 10 or 50 mM sodium
formate buffer at pH 3.2. However, FP-HM was not soluble under these conditions. All proteins
were soluble with 6 M GuHCl at either pH 3.2 (50 mM sodium formate) or pH 7.4 (10 mM
sodium phosphate). There was solubility either with or without 150 mM NaCl. At pH 7.4, there
was no appreciable solubility with [GuHCl] < 6 M.
3.3.3. Monomer or Hexamer and Not Trimer.
Oligomerization was probed by SEC for conditions that satisfied the above solubility criteria.
The typical protein loading was 1 mg/mL and there was ~10-fold dilution in the column. For a
particular buffer, the dominant oligomeric state was either monomer or hexamer and never
trimer. For example, for HP at pH 3.2 without GuHCl, the SEC profile is consistent with a
monomer (Figure 3.6A). This is significant as CD, calorimetric, and vesicle fusion studies of gp41
ectodomain constructs have often been done with protein at pH 3 (18,35,37). It has typically
been assumed that there are SHB trimers; however, the SEC only shows monomers. For 10 mg
HP/mL loading concentration, the SEC profile also shows a major fraction monomer with much
smaller fractions of trimers, hexamers, and larger aggregates (Figure 3.6B). SEC was attempted
for HM at pH 3.2 without GuHCl but the protein bound to the Superdex column material. For
buffer with 6 M GuHCl at either pH 3.2 or pH 7.4, both HP and HM show major fraction
hexamer with much smaller fractions assigned to dodecamers, 24-mers, and aggregates with
MW > 2 MDa (Figure 3.7). Although cell physiology does not include 6 M GuHCl, it is the only
condition we found that resulted in a defined small oligomer state. The hexamer is also the

81

dominant species when non-native protein tags are added to the ectodomain construct (38).
We therefore used 6 M GuHCl as one initial solubilization condition in subsequent experiments.
The major hexamer population and minor populations of larger oligomers likely represent a
thermodynamic equilibrium state. This was evidenced by collecting the hexamer fraction,
concentrating to 1 mg/mL, and then reloading this fraction for a second round of SEC. The
resultant SEC profiles for HP and HM are similar to those initially observed (Figure 3.9).

Figure 3.6. SEC A280 profiles for HP with (A) 1 mg/mL loading, Superdex 200 column and (B) 10
mg/mL loading, Superdex 75 column. The loading and running buffer was 50 mM sodium
formate pH 3.2, 150 mM NaCl, and 0.2 mM TCEP. The blue numbers are the MW standards in
kDa and their respective elution volumes are the downward blue arrows. There are
accompanying SEC profiles of the MW standards as well as plots of Kav vs log10(MW) (Chapter 2,
Figure 2.13).

82

Figure 3.7. SEC A280 profiles from a semi-preparative Superdex 200 column for HP and HM in
buffers containing 6 M GuHCl and 150 mM NaCl. The pH 3.2 buffer was 50 mM sodium formate
with 0.2 mM TCEP and the pH 7.4 buffer was 10 mM phosphate with 2 mM DTT. For each run,
the loading and running buffers were the same and the loading stock solution had a 1 mg/mL
protein concentration. The blue numbers are the MW standards in kDa and their respective
elution volumes are the downward blue arrows. The most prominent peaks in the profiles
correspond to hexamer masses and vertical dashed lines show the elution volume offset
between the HP and HM hexamers. Smaller peaks most consistent with dodecamer and 24-mer
masses are identified by shaded regions. For HM, there is also a small peak associated with the
Vo void volume, i.e. aggregates with MW  2 MDa.
83

Figure 3.8. Evidence for thermodynamic equilibrium between hexamers and larger oligomers.
SEC with A280 detection was run for HP and HM in buffer containing 50 mM sodium formate at
pH 3.2 with 150 mM NaCl, 6 M GuHCl, and 0.2 mM TCEP (Figure 3.7). The hexamer peak was
collected and loaded onto the Superdex 75 column for SEC with this same buffer. The loading
solution contained (A) 0.3 or (B) 0.2 mg protein/mL. Each blue number is the MW of a standard
in kDa and the blue downward arrows is its elution volume from Figure 2.13, Chapter 2.The
resultant profiles for (A) HP and (B) HM show a mixture of hexamers and larger oligomers
similar to the profiles of the initial run (Figure 3.7). This similarity supports this mixture as the
thermodynamic equilibrium state for the protein.
3.3.4. Hyperthermostable Hairpin Structure of the Monomer
CD spectra of HP and HM at pH 3.2 without GuHCl have the 208/222 nm minima characteristic
of  helical structure and the 222 values are consistent with dominant helical structure (Figure
3.10A). HP is predominantly a monomer in this buffer so the data support a highly helical
monomer. There is a small fraction of trimer for high SEC loading and the CD spectrum of this
84

fraction is also consistent with dominant helical structure. For HM, there was a small linear
decrease in |222| between 20 and 95 oC (Figure 3.10B) that is similar to the previously
observed temperature-dependence for HP and other large ectodomain constructs at pH 3. This
correlates with calorimetrically-determined Tm’s of ~110 oC (18).
CD spectra for the D632A and D632A/W628A mutants of HP provide additional support for the
helical monomer model (Figure 3.9C). In the atomic-resolution SHB trimer structure, D632 and
W574 form an inter-monomer CHR/NHR salt bridge and W628 and W571 form an intramonomer hydrophobic interaction (39). There is negligible difference between the WT and
D632A CD spectra whereas there is about two-times less magnitude molar ellipticity in the
D632A/W628A spectrum that is consistent with a significant loss in helicity. These data support
hairpin monomer structure of HP which is similar to a monomer unit of the SHB trimer
structure (Figure 3.17A). The Tm of ~110 oC corresponds to unfolding of hairpin monomer rather
SHB trimer.
For HP and HM in 6 M GuHCl at either pH 3.2 or pH 7.4, there is a 222 nm minimum consistent
with helical structure with significant absorption/interference below 215 nm from the GuHCl
(Figure 3.9D). The hexamer is the dominant oligomeric state under these conditions and may be
a dimer of two SHB trimers. For HP in 6 M GuHCl at pH 7 between 20 and 90 oC, there is a linear
decrease in |222| comparable to the figure 3.9B change without GuHCl. This supports
thermostability of the hairpin monomer structure within the hexamer.
The ligations are done in 8 M GuHCl at pH 7 and ambient temperature so it is interesting to
consider the structural properties in this condition. For HP in 8 M GuHCl at 20 oC, the |222| is
85

about 60% of the value in 6 M GuHCl. In addition, the |222| in 8 M GuHCl exhibits a small linear
decrease between 20 and 70 oC (Figure 2-7C in Matthew J. Nethercott’s Dissertation). These
data are consistent with a dimer of SHB trimers in 8 M GuHCl.

86

Figure 3.9. (A) CD spectra of HP and HM in pH 3.2 buffer. All spectra show 222 indicative of a
significant fraction of  helical structure. (B) 222 vs temperature for HM at pH 3.2. No unfolding
transition is observed. (C) CD spectra of WT and mutant HP at pH 3.2. For a SHB trimer, the
D632A mutation could disrupt the inter-monomer salt bridge with K574 and the W628A
mutation could disrupt the intra-monomer hydrophobic interaction with W571. More positive
CD signal for the double but not the single mutant is consistent with monomer rather than SHB
trimer HP at pH 3.2. (D) CD spectra of HP and HM in buffer with 6 M GuHCl. The spectra show
222 indicative of a significant fraction of  helical structure. The [protein]  15 M (~0.2
mg/mL), the pH 3.2 buffers contained 50 mM formate with 0.2 mM TCEP, and the pH 7.4
buffers contained 10 mM phosphate and 2 mM DTT.
87

3.3.5. Hairpin Monomer With MPER is Highly Fusogenic
Earlier work has shown that peptides corresponding to the FP and the MPER regions induce
vesicle fusion (27,29). For the present study, we investigated the effects of these regions as part
of the gp41 ectodomain including the hairpin region. The typical vesicle composition was
POPC:POPG:Chol (8:2:5) and reflected the major fraction PC, minor fraction negatively charged
lipid, and mole fraction Chol in the membranes of HIV and its host cells (40). Fusion was studied
at final pHs of 3.2 and 7.4 which was motivated by previous studies with HP showing much
greater fusion of negatively charged vesicles at pH 3.2 relative to pH 7.4 (41). A second vesicle
composition was POPC:DOTAP:Chol (8:2:5) and contained positively-charged DOTAP rather
than negatively charged POPG. Comparison of results for the two compositions and two pH’s
provides insight into the role of protein/vesicle electrostatics because the POPG and DOTAP are
respectively negatively and positively charged at both pH’s whereas the protein is positively
charged at pH 3.2 and negatively charged at pH 7.4.
HP and HM were solubilized as monomers at pH 3.2 and induced rapid fusion of negativelycharged POPG vesicles at this pH with low 1:700 protein:total lipid mole ratio (Figure 3.10A). At
pH 7.4, comparable fusion required an order-of-magnitude higher 1:75 ratio (Figure 3.10B). For
1:150 ratio, there was negligible fusion which is consistent with earlier work. For positivelycharged DOTAP vesicles, there was little fusion at pH 3.2, measurable fusion at pH 7.4, and
higher fusion at pH 9.0 (Figure 3.10C,D). The different pH dependences for POPG and DOTAP
vesicles likely reflect differences in protein/vesicle electrostatic energy, i.e. (1) attractive energy
between the positively-charged protein and negatively-charged POPG vesicles at low pH and

88

between the negatively-charged protein and positively-charged DOTAP vesicles at neutral and
high pH; and (2) repulsive energy between the negatively-charged protein and negativelycharged POPG vesicles at neutral pH and between the positively-charged protein and positivelycharged DOTAP vesicles at low pH. Attractive energy results in a higher fraction bound protein
and repulsive energy results in a lower fraction. In the spatially restricted environment of
HIV/host cell fusion, there is likely high protein/membrane binding that is similar to nearquantitative binding during the pH 3.2 vesicle fusion of POPG vesicles. Higher fusion extent for
POPG vesicles at low pH relative to DOTAP vesicles at neutral and high pH may be a
consequence of the different magnitudes of attractive electrostatic energy. This hypothesis is
evidenced by calculated hairpin charges of about +10 at pH 3.2, –2 at pH 7.4, and -4 at pH 9.0.
For many data, fusion was rapid and occurred during the ~5 s dead-time of the assay which is
consistent with earlier studies. Other studies have also shown that as the fraction of charged
lipid in the vesicle is decreased, the fusion rate decreases but the fusion extent increases where
the latter effect is probably due to reduced intervesicle electrostatic repulsion. Fusion is
enhanced with inclusion of the MPER in the construct as evidenced by the following differences
for HM relative to HP with POPG vesicles: (1) at pH 3.2, higher initial fusion rate; and (2) at pH
7.4, higher final fusion extent (Mf). For pH 3.2, the final [protein] = 0.2 M (~0.003 mg/mL) and
the hairpin monomer likely makes initial contact with the vesicle. HM-induced fusion occurs
within a few seconds, so the monomer is likely a fusion-active species. The enhanced fusion
with inclusion of the MPER is consistent with earlier observations of vesicle fusion induced by
MPER peptides.

89

Figure 3.10. Vesicle fusion at 37 oC. Protein was added just prior to the increase in fusion with
~5 s assay dead time. For panels A and B, the vesicles were negatively-charged and for panels C
and D, the vesicles were positively-charged. The calculated protein charge is +16, –2, and –4 at
respective pH values 3.2, 7.4, and 9.0. The protein:lipid mole ratios were: (A) 1:700, (B) 1:75,
and (C, D) 1:300, with Chol not included in the lipid quantity. The data overall support the
importance of attractive protein/vesicle attractive energy for fusion at low protein:lipid ratios.
The protein stock contained 20 M protein (~0.25 mg/L) in 10 mM formate at pH 3.2 with 0.2
mM TCEP which are condition for predominant monomeric protein. For panels A and B, [POPC]
= 120 M, [POPG] = 30 M, and [Chol] = 75 M, and for panels C and D, the vesicles contained
[POPC] = 120 M, [DOTAP] = 30 M, and [Chol] = 75 M. Vesicle buffers were 10 mM formate
at pH 3.2, 5 mM HEPES/10 mM MES at pH 7.4, and 10 mM Tris-HCl at pH 9.0.
90

3.3.6. Synergy of FP, Hairpin, and MPER With the Gp41 Copy Number of a Virion
The individual and collective contributions of the FP, hairpin, and MPER to ectodomain-induced
fusion were studied with HP, HM, and FP-HM solubilized as hexamers in 6 M GuHCl. The final
[GuHCl] = 40 mM and in the absence of protein, only modest fusion was observed at pH 3.2 (Mf
4%) and no fusion was observed at pH 7.4 (Figure 3.13). The probable reason for GuHCl to
induce fusion to some extent is due to reduction in electrostatic repulsion between the
negatively-charged vesicles due to the presence of bound GuH+ ions. Much greater fusion was
observed with protein (Figure 3.11). Relative to HP which lacked most of the MPER, inclusion of
the full MPER in HM resulted in higher Mf. Even greater Mf was induced for FP-HM which
contains both the FP and MPER. Similar quantitative binding was observed in the absence and
presence of the FP (41,42). Dose response was observed for HM and FP-HM (Figure 3.12). A
substantial Mf of ~15% was observed for FP-HM even at ultra-low protein:lipid = 1:4800 that
corresponds to ~15 proteins per ~100 nm diameter vesicle. This number is significant because it
is comparable to the ~30 gp41 molecules per virion. To our knowledge, this is the first
observation of rapid (~5s) ectodomain-mediated vesicle fusion at the protein copy number of a
virion. In some contrast, rapid FP or MPER peptide-induced vesicle fusion has typically been
observed at 500-1000 peptides per vesicle. Our result highlights the importance of the whole
ectodomain in fusion, including contributions from FP, hairpin, and MPER regions (43,44). The
fusogenicity of FP-HM is even higher than shown in Fig. 8 because our FP-HM stock contained
~40% HM impurity.

91

Figure 3.11. Vesicle fusion at 37 oC. A 7.5 L aliquot of protein was added just prior to the
increase in fusion and the final total volume was 1200 L. The POPC:POPG:Chol vesicle
composition and buffers were the same as Fig. 6. The protein stock buffer was 10 mM formate
at pH 3.2 with 6 M GuHCl and 0.2 mM TCEP and following addition of protein stock, the assay
buffer contained 40 mM GuHCl. For the pH 3.2 assays, the stock [protein] = 20 M (~0.25
mg/mL), and for the pH 7.4 assays, the stock [protein] = 160 M (~2 mg/mL). The
[protein]:[POPC+POPG] = 1:1200 for the pH 3.2 assays and 1:150 for the pH 7.4 assays.

92

Figure 3.12. Dose response of vesicle fusion induced by HM and FP-HM at pH 3.2. The assay
conditions were the same as Fig. 7A except that the protein stock concentrations were 20, 10,
and 5 M for [protein]:[total lipid] = 1:1200, 1:2400, and 1:4800, respectively.

Figure 3.13. Vesicle fusion induced by GuHCl at (A) pH 3.2 or (B) pH 7.4. A 7.5 L aliquot
containing 6 M GuHCl was added to a vesicle suspension with final total [GuHCl] = 40 mM in a
volume of 1200 L. The assay conditions were the same as Figure 3.7 except that there was no
protein in the aliquot that contained GuHCl.
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3.3.7. Folded Hairpin Ectodomain With MPER Binds to bNAbs.
The MPER region of gp41 includes the linear epitopes of several bNAbs and there is
consequently continued effort to develop a MPER-based immunogen as a HIV vaccine. HM or
similar folded hairpin molecules are appealing because of their high stability but there are
conflicting data about the extent to which they bind bNAbs. However, our Western blots show
reproducible binding of HM to the 4E10 bNAb (Figure 3.14A). HP lacks the 4E10 epitope and
serves as a negative control. Western blots also show that HM binds to the 2F5 bNAb more
strongly than HP which includes only part of the 2F5 epitope (Figures 3.14B and 3.1B). In the
Western blot approach, the protein is in 15% SDS prior to antibody binding. Although we do not
know HM structure in this condition, HM hairpin structure is hyperthermostable and a shorter
ectodomain construct without the MPER forms crystals in SDS with helical SHB structure (45). A
gel shift is not observed between HP and HM (Figures 3.5B and 3.14B) and could be the result
of folded rather than random coil structure. HM is predominantly monomeric in 15% SDS and
may therefore bind bNAb as a hairpin monomer. The blots also show minor dimer populations
binding to 2F5.

94

Figure 3.14. Western blots showing binding of HM to the 4E10 and 2F5 bNAbs and weaker
binding of HP to 2F5. The left-side arrows are MWs in kDa.
Binding to bNAbs was probed under physiological conditions using direct immunoprecipitation.
The protein stock solution contained either monomeric or hexameric protein and the binding
was done at pH 7.4 with [GuHCl] < 10 mM. For either initial oligomeric state, HM bound to 4E10
and 2F5 (Figure 3.15) whereas there was no binding to IgG (Figure 3.16). This is the antigenicity
needed for a vaccine immunogen. HP binds 2F5 weaker than HM which is consistent with an
incomplete epitope in HP.

95

Figure 3.15. SDS-PAGE of the immunoprecipitation of HP and HM with the 4E10 and 2F5 bNAbs.
There is stronger binding of HM to 4E10 and 2F5 and weaker binding of HP to 2F5. The protein
stock buffer contained 50 mM sodium formate at pH 3.2 with 150 mM NaCl. For the right three
lanes of the panel A gel, the buffer also contained 6 M GuHCl whereas for the left three lanes of
panel A, the GuHCl was absent. Binding was done in PBS at pH 7.4 and for the right lanes, the
diluted [GuHCl] = 40 mM. The center lane is the MW ladder in kDa.

96

Figure 3.16. SDS-PAGE of the immunoprecipitation of HP and HM with IgG. Protein binding to
IgG is not observed. The protein stock buffer contained 50 mM sodium formate at pH 3.2 with
150 mM NaCl. For the right two lanes of the gel, the buffer also contained 6 M GuHCl. The
antibody binding was done in PBS at pH 7.4 and for the right lanes of the gel, the diluted
[GuHCl] = 40 mM. The inner lanes are MW ladders in kDa.
3.4.

Discussion

Significant findings: The ectodomain of the HIV gp41 protein plays a major role in catalyzing
fusion between HIV and host cell membranes. The likely respective binding of the FP and MPER
to the host cell and viral membranes are probably part of the underlying physical basis for
catalysis. Although there has been significant functional and structural study of FP and MPER
peptides, there have been just a few investigations of these regions as part of the larger
ectodomain. The present work shows the significance of the FP, hairpin, and MPER regions for
ectodomain-induced vesicle fusion including fusion with ~15 proteins per vesicle which is

97

comparable to the gp41 copy number of the virion. For FP or MPER peptides, there has typically
only been appreciable rapid vesicle fusion at 500-1000 peptides per vesicle and to our
knowledge, ours is this first observation of rapid vesicle fusion at the virion gp41 copy number.
Detection of either predominant monomer or hexamer gp41 ectodomain rather than trimer
gp41 ectodomain was incorporated into the new HIV/host cell membrane fusion model III
(Figure 3.19). One significant feature is inclusion of monomer ectodomain rather than trimer
ectodomain PHIhairpin folding at step 3. The free energy of gp41 ectodomain folding is
coupled to free energy of formation of membrane intermediates. Our work may also aid HIV
vaccine development via an immunogen with a folded gp41 ectodomain including MPER. This
immunogen has the advantage of very high stability but there is literature disagreement about
ectodomain binding to bNAbs. We show bNAb binding to gp41 ectodomain protein initially
prepared as a monomer or hexamer.
SSNMR is a novel and important tool in recombinant protein (RP) production: One key result
that moved the work forward was SSNMR detection of HM in IBs at ~300 mg/L culture. The
bottleneck to purified protein was therefore IB solubilization rather than expression and
subsequent effort was focused on increasing solubilization. This result exemplifies how SSNMR
is a general method to quantitate RP yield without solubilization or purification. High
expression was obtained with common and inexpensive plasmids, E. coli strain, and growth and
expression conditions. IB solubilization rather than expression may therefore be a general
determinant of purified protein yield. Our yields were 15-50 mg/L and obtained with single-step
affinity purification which should be advantageous in immunogen production. Our approach
contrasts with earlier efforts that used protein fusion tags that had to be cleaved (46).
98

Monomer and hexamer rather than trimer gp41 ectodomain: For gp41 ectodomain constructs
lacking solubility tags, only constructs with short NHR and CHR segments and no FP or MPER
appear to form soluble trimers near pH 7 (47). For longer constructs that contain some or all of
the FP and/or the MPER, e.g. HP and HM, the present and earlier studies show most protein
forming large aggregates (MW>2 MDa) even in the presence of non-ionic detergents (48).
There is solubility near pH 3 and many biophysical measurements have been done at this pH.
High fractional helicity and Tm > 100 oC were considered strong evidence of SHB trimers.
One important contribution of the present study is SEC showing predominant monomer rather
than trimer gp41 ectodomain at pH 3 (Figure 3.5). Our SEC monomer result is consistent with
the SEC of large ectodomain constructs done by other groups (Figure 1A from Ref. 48 and
Figure S1 from Ref. 51) although this monomer interpretation was typically not made by the
authors of these papers. For these latter studies, the construct was either NHR+native
loop+CHR, FP+NHR+native loop+CHR+MPER+TM, or short NHR+short loop+short CHR.
The gp41 ectodomain monomer is highly helical and hyperthermostable. The most plausible
monomer structure is N-helix/180o turn/C-helix model (Figure 3.17A) similar to the monomer
unit of the high-resolution SHB trimer structures. The trimer is stabilized by several intermonomer NHR/NHR hydrophobic interactions and small reorientations of helices could reposition residues for favorable intra-monomer NHR/CHR interactions. A looser helical monomer
hairpin structure has been observed for a short NHR+loop+CHR construct in non-ionic
detergent but we favor the tight structure of figure 17A (49). Large ectodomain constructs like
HP and HM have Tm>100 oC either in the absence or presence of detergent and this high

99

thermostability likely reflects substantial interhelical contact in the folded monomer. The looser
structure of much shorter ectodomain constructs may be reflected in their typical Tm ≤ 70 oC.

Figure 3.17. Working structural models of the gp41 ectodomain with figure 3.1 color coding.
Monomeric HP is modeled as the hairpin structure of a monomer in the SHB. HM and FP-HM
hexamers are modeled as two SHB trimers that contact at their N-/C-terminal interfaces. This
model correlates with the observed intermolecular antiparallel  sheet structure of FPs.
Measurements of large ectodomain constructs at low pH should be interpreted in the context
of monomer protein. For example, the hyperthermostability detected by calorimetry and CD is
likely a consequence of intra-molecular NHR/CHR interaction rather than inter-molecular
NHR/NHR interaction. The stability of the ectodomain monomer would be consistent with an
important role for the monomer in fusion, discussed in a subsequent section and presented in
Figure 3.19. We also note that rapid vesicle fusion using stock protein at low pH is likely a
consequence of membrane perturbation by hairpin monomer rather than SHB trimer. This
includes fusion of negatively-charged vesicles at pH 3.2 (Figure 3.10A) and positively-charged
vesicles at pH 7.4 and 9.0 (Figure 3.10C,D).
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For 10 mg HP/mL SEC loading, there is a major monomer peak as well as minor trimer and
oligomer/aggregate peaks (Figure 3.9B). The normalized values of monomer:trimer:larger
oligomer integrations are: 0.72:0.18:0.10. These correspond to [monomer]  60 M and
[trimer]  5 M using the experimentally-determined ten-fold dilution in SEC. For the
association equilibrium, 3 monomer  trimer, the derived Ka  2107M –2, and corresponds to
equal mass concentrations of monomer and trimer for total HP of ~9 mg/mL (~90 mg/mL
loading in SEC). Earlier analytical ultracentrifugation (AUC) data have been analyzed to yield
Ka51111M

–2

(14,50,51). We do not understand the discrepancy between the SEC and AUC

analyses.
Protein hexamers were observed in 6 M GuHCl at either pH 3.2 or 7.4 (Figure 3.9). The data
show the hexamer as the fundamental unit of oligomerization rather than the trimer, e.g.
dodecamers were observed but not nonamers. A reasonable model for a hexamer is a dimer of
SHB trimers with inter-trimer contact between the MPERs (Figure 3.17B). This model is
consistent with the observed helical structure in the hexamer and with Tm > 100 oC. This model
is also consistent with some of the crystal packing of SHB trimers (39). In addition, SSNMR
studies of FP structure in the membrane-associated gp41 ectodomain show an oligomeric FP 
sheet with antiparallel but not parallel arrangement of the FP strands (24). The antiparallel
arrangement would be consistent with end-on arrangement of two SHB trimers (Figure 3.17C).
It is important to consider the impact that GuHCl may have on the ectodomain structure as
GuHCl often denatures proteins. We think denaturation is unlikely for the ectodomain because
there is substantial helical structure and Tm > 100 oC. In addition, to our knowledge, it is very

101

unusual that denatured protein would have a well-defined oligomeric state other than
monomer. A control experiment was performed by running a size exclusion chromatography
experiment of protein GB1 in the presence of 6M GuHCl at low pH 3.2 and physiologic pH 7.4.
The protein GB1 construct which has been studied has a MW ~6.2 KDa. Previous spectroscopic
studies have showed the complete unfolding of protein GB1 in the presence of 6M GuHCl (52).
Consistent with this result, we have obtained a clear monomer peak of the protein GB1 in the
presence of 6M GuHCl with the MW equal to ~6.3 kDa and ~9 kDa at pH 3.2 and 7.4
respectively (Figure 3.18). There was no other peak showing the presence of higher order
oligomer. The hexamer is also the dominant oligomeric state of a modified ectodomain
construct in physiologic solution, i.e. pH 7.4 without any GuHCl (38). This Soc-gp41ectoM-Fd
construct includes a central gp41ectoM region which is very similar to FP-HM as well as a ~9
kDa non-native Soc protein N-terminal of the FP and a ~2 kDa Fd non-native protein C-terminal
of the MPER. The Soc and Fd proteins were chosen because they form homotrimers in
physiological solution. However, there was only minor trimer population of Soc-gp41ectoM-Fd
in physiologic solution. Instead, the hexamer was dominant with additional smaller populations
of two larger oligomers. The overall oligomer distribution of Soc-gp41ectoM-Fd in physiologic
solution is very similar to that of HP and HM in 6 M GuHCl with dominant hexamer and two
minor higher-order oligomers that are reasonably assigned to dodecamer and 24-mer. It is
likely that there are similar hexamer structures of HP, HM, and Soc-gp41ectoM-Fd.

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Figure 3.18. The monomeric elution peak of protein GB1 in PBS in the presence of 6M GuHCl at
(A) pH 3.2 and (B) pH 7.4. The elution volume corresponds to the molecular weight of ~6.3 kDa
and ~9 kDa at pH 3.2 and 7.4 respectively. The size exclusion chromatography was done at
Superdex 75 column. The blue arrows show the elution volumes of MW standards with the
MWs given in kDa.
At neutral pH, large ectodomain constructs form >2 MDa aggregates whereas at low pH,
monomers predominate for <10 mg protein/mL and there is significant trimer population with
SHB structure for >50 mg protein/mL. The pH-dependence of oligomeric state is observed with
or without non-ionic detergent so our explanatory model is based on relative magnitudes of
protein electrostatic vs hydrophobic effects. Trimer stabilization is primarily due to hydrophobic
interaction between the three NHR helices on the SHB interior. At low pH, there is a calculated
charge of ~+10 for the hairpin region of the ectodomain monomer. We posit that monomer
rather than oligomers are favored at low pH because inter-molecular electrostatic repulsion
103

overwhelms the hydrophobic interaction. At neutral pH, the calculated charge is –2 and
hydrophobic interaction is dominant. The resulting aggregates may be composed of SHB
trimers.
For solutions containing GuHCl at both low and neutral pH, hexamers predominate, i.e. addition
of

GuHCl

induces

monomerhexamer

oligomerization

at

low

pH

and

induces

aggregatehexamer breakup at neutral pH. To our knowledge, the specific interaction of
positively-charged GuH+ with protein molecules is not understood (53). For the ectodomain
protein, we posit that the hexamer is formed from two SHB trimers (Figure 3.17B,C) and that
GuH+ interacts with COO– groups on the SHB surfaces. The -COO– rather than the -COOH state is
favored at both pH’s because of attractive interaction with GuH+. At low pH, this reduces the
positive charge of the protein so that the SHB trimer does not dissociate due to electrostatic
repulsion between monomers. Hexamers form because of favorable hydrophobic interaction
between the FP/MPER end of one SHB trimer and the FP/MPER end of a second trimer.
Aggregation beyond hexamers is disfavored because this aggregation would reduce exposed
protein surface area and therefore the number of GuH+/COO– contacts. As noted above, SOCgp41ectoM-Fd forms a hexamer in the absence of GuHCl and we posit this is also two SHB
trimers. Further aggregation is disfavored because of the solubility of the SOC and FD proteins
and perhaps because of their steric bulk.
Correlation of vesicle fusion with HIV/cell fusion: One significant result is observation of vesicle
fusion for ~15 FP-HM per vesicle (Figure 3.12B) which is comparable to the gp41 copy number
of ~30 in the virion. To our knowledge, FP-HM thus provides the first example for which

104

protein-induced vesicle fusion might reasonably be directly compared to viral fusion. FP-HM is
more fusogenic than shorter ectodomain constructs like HP which lacks the FP and MPER, and
also more fusogenic than isolated FP or MPER peptides. This potent fusogenicity highlights the
importance of the full ectodomain in fusion, with inclusion of the N-terminal FP, hairpin region,
and C-terminal MPER. During HIV/cell fusion, the FP and MPER likely bind to the cell and HIV
membranes, respectively, and higher fusion for FP-HM relative to HM supports synergy
between these two regions, likely via membrane perturbations. Vesicle fusion is probably a
better model of the earlier hemifusion (lipid mixing) step of viral fusion than the final fusion
pore expansion step. Pore formation and expansion in vesicle fusion is less regulated than in
HIV/cell fusion and leakage of contents out of the vesicles occurs much faster than mixing of
contents between vesicles.
For conditions with attractive protein/vesicle electrostatic energy, protein/vesicle binding is
quantitative and FP-independent. The observed FP-dependent fusogenicity is therefore not
reasonably ascribed to FP-dependent binding. Protein binding is described by the equation fm =
(Km[L])/{1+(Km[L])} where fm  fraction bound-protein, Km  equilibrium constant derived
from Gm  binding free-energy, and L  lipid. For our assays, [L] = 1.510–4 M and
protein/vesicle binding is quantitative (fm = 1.0) when Km > 105 M. The Gm = Gelec + Ghydro and
corresponding Km = Kelec  Khydro describe contributions from electrostatic and hydrophobic
interactions. The FP is hydrophobic but lacks ionizable residues so it affects Ghydro but not Gelec.
For negatively-charged vesicles at pH 3 and positively-charged vesicles at pH 9, the calculated
Kelec > 105 M and corresponds to quantitative binding of all proteins, which has also been
experimentally-observed. For conditions with much smaller Kelec, such as negatively-charged
105

vesicles at neutral pH, binding may be FP-dependent and changes in fusogenicity could be
related to binding differences.
High fusion by FP-HM requires a pH for which there is attractive protein/vesicle electrostatic
energy with resultant quantitative binding to the vesicles (Figure 3.10). Attractive electrostatic
energy is not a requirement for fusion by the ectodomain of full-length gp41 in the virion, likely
because the ectodomain is restricted to the space between the viral and host cell membranes.
This spatial confinement favors protein binding to the membranes even without electrostatic
attraction.
For the present study, a 0.2 fraction charged lipid was used which is larger than the typical
fraction in the cell membrane. However, a recent study showed efficient vesicle fusion with
much smaller fractions charged lipid (42). As the fraction charged lipid is decreased, there is a
corresponding increase in fusion extent that is likely due to decreased intervesicle electrostatic
repulsion. Some charged lipid is needed for quantitative protein binding to the vesicles and
consequently fusion. As one example, HP at protein:lipid = 1:700 (~100 HP molecules/vesicle)
induced ~40% fusion of vesicles containing only 0.02 fraction negatively-charged
phosphatidylserine lipid. These results, along with the higher fusogenicity of FP-HM relative to
HP (Figure 3.11A), support correlation between FP-HM induced vesicle fusion and hemifusion
between the HIV and cell membranes mediated by the viral ectodomain.
New fusion model with gp41 monomer and hexamer ectodomain: As noted in the Introduction,
models for gp160-induced membrane fusion have typically considered changes in structure of a
gp160 trimer that are temporally and energetically coupled to changes in the topologies of the
106

HIV and host cell membranes. Models I and II of the Introduction share the feature of trimeric
ectodomain gp41 throughout fusion. For model I, concerted PHISHB trimer folding is coupled
to subsequent hemifusion. However, this is inconsistent with fusion inhibition by CHR+MPER
peptides up to the final pore expansion step and the corollary assumption of peptide binding to
the PHI trimer but not the SHB trimer. Model II delays the PHISHB trimer folding until the
pore expansion step which suggests that the SHB is only associated with fused membrane
stabilization and fusion arrest. The new model III incorporates asynchronous ectodomain
monomer PHIhairpin folding followed by changes in membrane topology leading to
hemifusion (Figure 3.19). The steps of membrane fusion led by gp41 according to the proposed
new model are as follows - (1) the gp120 and gp41 are non-covalently associated as a trimer
structure. At this stage the FP is expected to be inside the hydrophobic core of gp120 and gp41
trimer structure, as FP is a very hydrophobic domain. (2a&b) After the gp120 binds to the host
cell receptors and coreceptors, gp120 moves away, and gp41 is exposed. The FP comes very
close to the host membrane, which further helps FP to insert into the host membrane and
stabilize. However, this close proximity of the host and viral membrane is only possible if the
gp120 is still bound to the gp41 while being attached to the host cell receptors and coreceptors.
On the other hand this attachment of gp120 to the gp41 is very unlikely as in the presence of
gp120, the folding of gp41 into hairpin conformation and subsequent SHB formation would not
be possible due to steric hindrance. (3-5) There is subsequent ectodomain monomer hairpin
assembly into SHB trimers and (6-7) final assembly of two trimers into a hexamer with an
antiparallel FP  sheet. During viral fusion, the N-terminal FP is likely attached to the cell
membrane and the C-terminal TM is in the viral membrane. In our view, these membrane
107

topologies are more reasonably maintained with asynchronous folding of individual monomer
ectodomains than with concerted folding of the three ectodomains of the trimer. Asynchronous
ectodomain monomer folding to a hairpin is also consistent with the hyperthermostability (Tm 
110 oC) of the monomer hairpin.

108

Figure 3.19. Model for membrane fusion that includes the gp41 ectodomain monomer and
hexamer. The different regions of gp41 are color coded according to figure 3.1 and the TM and
endodomains are not shown. One of the monomers is not displayed in steps 3-5. The initial
gp41 structure of step 1 is based on the ~5 Å gp140 structures and the final SHB structure of
step 7 is based on high-resolution structures.

109

There is evidence supporting the involvement of monomer hairpin gp41 ectodomain at some
stage of HIV/host cell fusion. Such fusion has typically been assayed using the surrogate system
of fusion between cells expressing gp160 and cells expressing CD4 and co-receptor proteins.
Fusion is quantitated using the number of syncytia (fused cell aggregates). There is typically
dramatic reduction in fusion with mutations impacting intra-monomer CHR/NHR interactions,
e.g. no syncytia are formed with the W571R mutation (54). Membrane fusion and HIV infection
are inhibited by the clinically prescribed CHR+MPER enfuvirtide therapeutic which could act by
binding to exposed NHR surfaces in the PHI or hairpin ectodomain monomer but would not
bind to the SHB trimer for which the three NHR helices are on the bundle interior (55). The
physiological relevance of the gp41 ectodomain monomer is also supported by observation of
monomer rather than trimer WT gp140 protein (8). Gp140 is a non-covalent complex between
the HIV gp120 protein and the ectodomain of gp41 but lacks the TM and intraviral regions of
gp41.
A role for hexameric gp41 in HIV/cell fusion is supported by the trans-dominant effect of the
V513E mutation in the FP region of gp41 on gp160-mediated membrane fusion and HIV
infection (56). These data have been modeled using a requirement of multiple gp160 trimers
for fusion and infection (26). A membrane-associated gp41 hexamer is consistent with the
antiparallel  sheet FP structure in the membrane-associated gp41 ectodomain observed by
SSNMR (24). Such structure is consistent with interleaved FP strands from two trimers (Figure
3.17C).

110

After submission of the present paper, another paper was published showing that a shorter
NHR+loop+CHR gp41 ectodomain construct is monomeric at low pH in non-ionic detergent (49).
This result is consistent with our observations for the larger HP and HM constructs. We also
observed that pH rather than detergent is the critical parameter for solubility. In the absence of
GuHCl, both the HP and HM constructs are soluble at low pH and insoluble at neutral pH and
these results are independent of the presence or absence of detergent. The short construct in
detergent at low pH has a loose hairpin structure with non-interacting NHR and CHR helices
which lie on the micelle surface. The low pH micelle location is consistent with our observations
of protein-to-lipid headgroup contact for a large, membrane-associated ectodomain construct.
At low pH, NHR+CHR backbone

13

CO-to-lipid 31P distances of 8-9 Å were observed via SSNMR

spectroscopy (42). At neutral pH, there was no detectable contact which corresponds to 13CO31

P distances >12 Å even though the protein remained membrane-bound via the FP region. The

pH-dependence of the protein-to-lipid distances is consistent with the attractive
protein/membrane electrostatic energy at low pH and repulsive energy at neutral pH.
Although not included in the gp41 constructs of the present study, the TM domain is important
for anchoring gp41 in the viral membrane and is likely also important in fusion pore formation
(57). The TM sequence is conserved across clades of HIV and this conservation may be needed
for efficient trafficking of gp160 to the infected cell membrane surface prior to viral budding
(58). There are moderate effects on fusion and infection with some mutations in the TM region
(59,60). It would be interesting in the future to study the oligomerization and fusion activity of
gp41 constructs that include the TM domain. SEC of a full ectodomain+TM construct showed
predominant monomers at pH 4 in detergent which is consistent with the SEC for ectodomain
111

without TM (51). There was little contact between the FP and TM regions for the large
construct in detergent but some contact has been observed between synthesized FP peptide
and TM peptide in membranes (61).

112

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113

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119

CHAPTER 4
Construction of HIV-1 Gp41 Pre-Hairpin Intermediate

120

4.1.

Specific Aims

The long-term goals of the proposed research were to elucidate the structure of the
intermediate state (Pre-Hairpin Intermediate) of envelope fusion protein gp41 and
development of the structure-function model for the whole ectodomain region of the viral
fusion protein. This study would help us to understand the mechanism of fusion of viral and
host cell membranes and put a step forward towards the development of novel antiviral
therapeutics targeting the fusion process.
The specific aims of this project include:
(1)

Mutation of Hairpin region to mimic Pre-Hairpin Intermediate (PHI) state.

(2)

Determination of the secondary structure of the wild type and mutated protein

constructs by circular dichroism.
4.2.

Introduction

During the fusion process of the viral and host cell membrane, it is proposed that the viral
protein gp41 undergoes three states sequentially: (a) Pre-fusion native state, (b) Pre-Hairpin
Intermediate state (PHI), and (c) Final state (Hairpin) (1) (Figure 4.1).

121

Figure 4.1. HIV fusion model. (a)Native state of envelope protein before fusion, (b) Pre-Hairpin
intermediate of gp41, and (c) Final Hairpin state of gp41 (2).
Previously, biophysical studies were done to understand which conformation is involved in the
membrane fusion. Lipid mixing assays (assays to measure mixing of membrane lipids) of large
unilamellar vesicles was performed to assess the extent of vesicle fusion induced by FP, FPNHR, FP-Hairpin (FP-HP), and Hairpin. The experimental conditions were low pH 3.0 and
physiologic pH 7.4. The vesicles are negatively charged at both pH 3.0 and 7.4. The protein
constructs are positively charged at both the pHs, however, the magnitude of charge is greater
at pH 3.0 than compared to pH 7.4. It was shown that the FP-HP does not induce significant
vesicle fusion, whereas, FP-NHR construct which is a model of N-terminal PHI induces rapid and
extensive vesicle fusion. It has also been shown that the FP-HP and Hairpin construct inhibits
vesicle fusion when induced by FP-NHR construct (1).
X-ray crystallography studies have been performed to solve the structure of the Hairpin and
Hairpin-MPER in its probable final gp41 ectodomain structure during fusion (1,3-5). The trimer
of Hairpin structure assembles into a six-helix bundle (SHB). There are recent ~5Å structures of
gp140 trimers that possibly represent the protein state prior to membrane fusion (6-8). The
122

trimer gp140 is stabilized by mutations and ectodomain cross-linking between gp120-gp41
(9,10). The structure shows loose bundle of three parallel NHR helices and three CHR helices
forming a tripod with an angle of 70° between the helices. In this gp140 structure, the FP and
MPER are not included.

The melting temperature (Tm) of only the NHR-CHR shorter

ectodomain construct is ~70 °C whereas the Tm of longer ectodomain constructs is ~110 °C
(2,11). This thermal stability leads to propose SHB as the final stable structure of gp41
ectodomain during fusion. PHI state of gp41 ectodomain has been proposed as fully extended
(NHR helix/0° -turn/CHR helix) with separate NHR and CHR trimer helical bundles rather than
hairpin conformation. To our knowledge, there is only functional evidence for the PHI state of
gp41. The presence of this intermediate is supported by inhibition of membrane fusion and HIV
infection with NHR and CHR+MPER peptides (12,13). A proposed mechanism of inhibition is
tight binding of the peptide to the PHI structure with consequent prevention of formation of
the SHB structure.
4.3.

Approach

4.3.1. Mutagenesis of CHR
Crystal structure of NHR-CHR peptides shows a trimeric assembly where NHR and CHR region of
one monomer are oriented in antiparallel manner. This six-helix bundle formed by the NHR and
CHR regions from three monomers is stabilized mainly due to hydrophobic interactions (3). The
trimeric NHR assembly is formed by hydrophobic interaction in the “a” and “d” positions as
shown in figure 4.2. The residues at “a” and “d” positions of CHR interact with the “e” and “g”
residues of the neighboring NHRs (Figure 4.2). W628, W631 of the CHR interacts with W571 of

123

the NHR. Similarly, I635 of CHR has hydrophobic contact with L565 and L568 of NHR. D632 of
CHR forms a conserved salt bridge with K574 of NHR.
Broadly neutralizing antibodies are capable of blocking different strains of virus from infecting
target cells. 2F5 and 4E10 are the two broadly neutralizing antibodies that bind to the MPER
only in the trimer state of gp41 with MPER fully exposed (14). Another structural study of
binding of broadly neutralizing antibodies to MPER show helical structure of peptide MPER as
prerequisite for antibody binding (15,16).
It was hypothesized that the PHI state could be obtained by disrupting the interactions
between the CHR and NHR. Mutation of W628A and W631R in the CHR might result in the
disruption of the hydrophobic interactions. The salt bridge between D632 and K574 can be
disrupted by mutating to D632A.

124

Figure 4.2. Helical wheel representation of NHR and CHR. Three NHR helices and one
CHR helix are represented as helical wheel projections. It is an end-on-view of the NHRCHR complex looking down the axis of the trimer. The NHR helices interact with other
NHR helices at the “a” and “d” positions (colored orange). Positions of the NHR and the
CHR helices that occupy the interhelical space between two NHR helices and one CHR
helix are shaded yellow (3). The residues which interact among themselves are colored
accordingly
4.3.2. Verification of Formation of Pre-Hairpin Intermediate
The Hairpin and the FP-HP are highly thermostable with melting temperature at 112°C (2).
Circular Dichroism (CD) spectra of Hairpin (HP) in aqueous solution show 100% helicity for the
peptide (Figure 4.3). CD is a very useful spectroscopic tool to verify global secondary structure
125

of large biological molecules. In this technique the difference in absorption of left and right
circularly polarized light by chiral molecules is recorded and plotted against the wavelength.
Percent helicity can be calculated from the mean residue molar ellipticity (θ222) at wavelength
222nm (minima). θ222 of -33,000 deg cm2 dmol-1 at wavelength 222nm is considered to be 100%
(17). CD signal at 222 nm can be measured as a function of temperature and thermal melts can
be performed at 2 °C steps between 20 °C – 100 °C.

Figure 4.3. (Left) Far-UV CD analysis of Hairpin showing 100% helicity with the mean residue
molar ellipticity at 222nm (θ222) is -33,000 deg cm2 dmol-1. For inset, abscissa is the temperature
scale in °C, and the ordinate is θ222, showing the thermal stability of Hairpin with temperature.
(Right) Thermal stability of Hairpin () and N70 (+). The melting temperature of N70 is 45 °C (1).
Our goal was to have the PHI state such that the NHR region is undisturbed, forming the trimer,
and the CHR is disturbed by mutation and unable to interact with NHR. The mean residue molar
ellipticity of Hairpin at 222nm (θ222nm) is -33,000 deg cm2 dmol-1. θ222nm for an unfolded protein
is -5000 deg cm2 dmol-1 (1). There are 47 residues in the NHR, 6 residues in the loop region, and
39 residues in the CHR. Therefore, it is expected that the PHI should have θ 222nm value as ~ 126

19,000 deg cm2 dmol-1. The melting temperature (Tm) for N70 (FP-NHR) is 45 °C (1). Therefore,
the proposed PHI should also have Tm at ~ 45 °C.
4.4.

Materials and Method

4.4.1. Construction of Mutant HP and HM
The mutant HP and HM constructs were constructed by site directed mutagenesis. The
mutations done were as follows – (a) D632A, (b) W628A, and (c) W631R. All the three
mutations were done separately on HP and HM. Mutants M1 comprised of mutation D632A,
M2 comprised of mutations D632A and W628A, and M3 comprised of D632A, W628A, and
W631R.
Primers were designed based on the required mutations. The template plasmids were
extracted from overnight grown cells. PCR was conducted using HP or HM template DNA and
designed primers. The DNA sequence after mutation is shown in Figure 4.4.

127

HP M3
CTLTVQARQL

LSGIVQQQNN

RGGAMERARE INNYTSLIHS

LLRAIEAQQH

LIEESQNQQE

LLQLTVWGIK

KNEQELLELD

QLQARILSGG

KWHHHHHH

HP WT DNA Sequence
tgc acg ctg acg gta cag gcc aga caa tta ttg tct ggt ata gtg cag cag cag aac aat ttg ctg agg gct
att gag gcg caa cag cat ctg ttg caa ctc aca gtc tgg ggc atc aag cag ctc cag gca aga atc ctg tct
ggt ggc cgt ggc ggt tgg atg gag tgg gac aga gaa att aac aat tac aca agc tta ata cac tcc tta att
gaa gaa tcg caa aac cag caa gaa aag aat gaa caa gaa tta ttg gaa tta gat aaa tgg cat cac cat cac
cat cac tga
HPM3 DNA Sequence
tgc acg ctg acg gta cag gcc aga caa tta ttg tct ggt ata gtg cag cag cag aac aat ttg ctg agg gct
att gag gcg caa cag cat ctg ttg caa ctc aca gtc tgg ggc atc aag cag ctc cag gca aga atc ctg tct
ggt ggc cgt ggc ggt gcg atg gag cgg gcc aga gaa att aac aat tac aca agc tta ata cac tcc tta att
gaa gaa tcg caa aac cag caa gaa aag aat gaa caa gaa tta ttg gaa tta gat aaa tgg cat cac cat cac
cat cac tga
Figure 4.4. Amino acid sequence of HP protein construct, DNA sequence of HP WT and HP M3.
4.4.2. Experimental Procedure
The mutated Hairpin (D632A) with His-tag was expressed in BL21 (DE3) competent cells.
When proteins of one biological system are expressed in other biological (e.g., bacterial)
128

system, within the cell they sometimes exist as insoluble aggregates, known as inclusion bodies.
Purification of the Hairpin-His (D632A) protein was tried by sonicating the cells and pelleting
down the inclusion bodies along with cell debris. The pellet containing the inclusion bodies as
well as cell debris was sonicated again in the presence of (a) 8M urea or (b) 6M GuHCl and
centrifuged. Both urea and GuHCl could efficiently solubilize HP mutant containing inclusion
body. The supernatant containing the solubilized protein was collected. Purification was done
by metal affinity chromatography. All the steps in purification were done at pH 7.4 and at room
temperature.
Purification of HM mutants was done in similar process as HP mutants, however, only GuHCl
solubilized the protein containing inclusion bodies most efficiently. After the purification, the
required proteins were dialyzed against sodium formate buffer containing 0.2 mM TCEP and at
pH 3.2. All the mutants’ i. e. M1, M2 and M3 of both HP and HM were soluble at low pH 3.2.
The secondary structure of the soluble protein constructs were measured by performing
circular dichroism (CD). The CD experiments were done at pH 3.2 and 20 °C. The thermal
melting of the protein constructs were measured within the temperature range of 20 – 96 °C.
All the observations were subtracted by the reading observed by buffer only.
4.5.

Results

In the gp41 HP or HM construct, D632 in the CHR forms a salt bridge with K574 in the NHR
(Figure 4.5). The mutation of D632A breaks the salt bridge. There is a hydrophobic interaction
between W571 in NHR and W628 and W631 in CHR (Figure 4.5). Mutation of W628A and

129

W631R breaks the hydrophobic interaction. The insertion of charged residue R631 further
disturbs the hydrophobic environment.

Figure 4.5. The SHB trimer of gp41 comprising of inner core NHR and outer CHR. Each monomer
is colored differently. Top, residues shown forms intermonomer salt bridge, and bottom, shows
residues forming hydrophobic interaction between intramonomer strands.
The secondary structure of wild type (WT) HP or HM (Figures 4.6 and 4.8 respectively) showed
~100% helicity. The secondary structure of HP M1 shows similar helicity as HPWT (Figure 4.6).
130

However, the helicity is reduced by ~50% i.e. θ222 is ~ -20000 deg cm2 dmol-1with the second
mutation (W628A) (Figure 4.7). In case of HP M3 or HM M3, the percentage helicity remains
almost same as HP M2 (figure 7, 8, and 6 respectively). To understand the thermal stability of
all the three mutants, thermal melt experiments were performed. It was observed that the
melting temperature did not change much compared to the WT protein constructs (Figure 4.9).
After the unfolding of the protein by thermal melt experiments, the protein sample was
allowed to refold at ambient temperature for 2 hours. The extent of refolding was measured by
measuring the mean residue molar ellipticity at 20 °C. It was observed that HM WT and HM M3
both refolded after thermal denaturation.

131

Figure 4.6. CD spectra of WT and mutant HP at pH 3.2. For a SHB trimer, the D632A mutation
disrupts the inter-monomer salt bridge with K574 and the W628A mutation disrupts the intramonomer hydrophobic interaction with W571. The [protein]  15 M (~0.2 mg/mL), the pH 3.2
buffers contained 50 mM formate with 0.2 mM TCEP.

132

Figure 4.7. CD spectra of HP WT and HP M3 at pH 3.2. W631R mutation disrupt the intramonomer hydrophobic interaction with W571. The [protein]  10 M (~0.2 mg/mL), the pH 3.2
buffers contained 50 mM formate with 0.2 mM TCEP.

133

Figure 4.8. CD spectra of HM WT and HM M3 at pH 3.2. For a SHB trimer, W631R mutation
along with W628A disrupts the intra-monomer hydrophobic interaction with W571. The
[protein]  10 M (~0.2 mg/mL), the pH 3.2 buffers contained 50 mM formate with 0.2 mM
TCEP.

134

Figure 4.9. Estimation of thermal stability of mutant HM. Left, measurement of thermal
unfolding of HM WT and HM M3 with mean residue molar ellipticity as a function of
temperature at 222nm and pH 3.2, and right, CD spectra of thermally denatured protein after
refolding.
4.6.

Discussion

Based on the fusion model, it is proposed that the gp41 exists as trimer in the PHI state and
forms a six-helix bundle structure at the end of the fusion process. The crystal structure of gp41
comprising of the NHR and CHR shows trimer structure (5). However, the mutations we
performed to disrupt the NHR-CHR interactions revealed that gp41 NHR-short loop-CHR exists
as folded monomer structure. The mutation M1 (D632A) disrupts the inter-monomer saltbridge with K574 and mutations M2 (W628A and D632A) disrupts the intra-monomer
hydrophobic interaction with W571. The D632A (M1) did not change the secondary structure of
HP whereas there was decrease in α-helicity in the mutant M2 (Figure 4.6). This indicates that
there is a change in secondary structure by disruption of intra-monomer interaction whereas
no change by breaking of salt bridge between D632 in CHR and K574 in NHR of two individual
135

monomers. To add to this, we also verified the Tm of HM M3. It was observed that there was no
change in the melting point of M3 as compared with wild type HM. Even though we had
achieved the expected θ222 value ~-20000 deg cm2 dmol-1, we were unable to reach the desired
Tm value of the protein constructs. Unfolding of HP or HM (Wild type) to M3 does not
contribute to the lowering of melting point.
4.7.

Future Work

Our goal was to attain the gp41 ectodomain in the PHI state which would possess low helicity
and low Tm. We propose that mutations of few more residues in the CHR would disturb the
NHR-CHR interaction. Q653 of CHR has intramolecular hydrogen bond with Q551 of the NHR
within the gp41 monomer. Q653 also forms an intermolecular hydrogen bond with the
backbone carbonyl oxygen of V549 of NHR (3). The hydrophobic interactions between (a) H564
and Y638 and (b) L565 and I635 are formed between intra-monomer NHR-CHR. These intramonomer NHR-CHR interactions along with previously mentioned hydrophobic interaction
(figure 5 bottom) are near the loop region. We propose mutation of Q653A, I635K, and Y638K
would disrupt the interaction between NHR and CHR.

136

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Constructs†. Biochemistry 48, 2714–2722

2.

Sackett, K., Nethercott, M. J., Epand, R. F., Epand, R. M., Kindra, D. R., Shai, Y., and Weliky, D. P.
(2010) Comparative Analysis of Membrane-Associated Fusion Peptide Secondary Structure and
Lipid Mixing Function of HIV gp41 Constructs that Model the Early Pre-Hairpin Intermediate and
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3.

Chan, D. C., Fass, D., Berger, J. M., and Kim, P. S. (1997) Core structure of gp41 from the HIV
envelope glycoprotein. Cell 89, 263-273

4.

Weissenhorn, W., Dessen, A., Harrison, S. C., Skehel, J. J., and Wiley, D. C. (1997) Atomic
structure of the ectodomain from HIV-1 gp41. Nature 387, 426-430

5.

Shi, W., Bohon, J., Han, D. P., Habte, H., Qin, Y., Cho, M. W., and Chance, M. R. (2010) Structural
Characterization of HIV gp41 with the Membrane-proximal External Region*. Journal of
Biological Chemistry 285, 24290–24298

6.

Bartesaghi, A., Merk, A., Borgnia, M. J., Milne, J. L. S., and Subramaniam, S. (2013) Prefusion
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Cleaved HIV-1 Envelope Trimer. Science 342, 1477-1483

8.

Lyumkis, D., Julien, J. P., de Val, N., Cupo, A., Potter, C. S., Klasse, P. J., Burton, D. R., Sanders, R.
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and conformational properties of a proteolytically mature, disulfide-stabilized human
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10.

Sanders, R. W., Vesanen, M., Schuelke, N., Master, A., Schiffner, L., Kalyanaraman, R., Paluch,
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Lu, M., Ji, H., and Shen, S. (1999) Subdomain folding and biological activity of the core structure
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Markosyan, R. M., Cohen, F. S., and Melikyan, G. B. (2003) HIV-1 Envelope Proteins Complete
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Frey, G., Peng, H., Rits-Volloch, S., Morelli, M., Cheng, Y., and Chen, B. (2008) A fusionintermediate state of HIV-1 gp41 targeted by broadly neutralizing antibodies. PNAS 105, 3739–
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139

CHAPTER 5
Future Work

140

Previous solid state NMR study of longer ectodomain (FP-HP) of gp41 by our group showed
antiparallel beta sheet arrangement of FP domain located in the membrane (1). Our study of
gp41 ectodomain reveals existence of the ectodomain in monomeric and hexameric state
(explained in Chapter 3). We have also studied the functional significance of the oligomeric
gp41 ectodomains. Based on the mutational studies and thermal stability of gp41 ectodomain,
it has been proposed that monomer gp41 ectodomain forms a hairpin conformation in aqueous
solution at low pH. It is not known whether gp41 ectodomain forms monomer or higher
oligomer in membrane environment.
Therefore, in future studies could be done to determine the oligomeric state of whole gp41
ectodomain construct incorporated in lipid membrane (vesicles). A qualitative experiment
could be done by 20% 13C labeled and uniformly 2H labeled protein constructs incorporated in
membrane and measuring the dephasing between

13

C-2H by REDOR solid state NMR

experiment. It is expected to observe dephasing if there is any oligomerization and no
dephasing for monomeric state. However, by this experiment we could only differentiate
between monomer and any oligomer formation. We would not be able to identify between
types of oligomers formed by the gp41 ectodomain protein construct incorporated in
membrane. After, the determination of the probable oligomeric state of the ectodomain
protein construct, solid state NMR studies could be done to investigate the conformation of
monomer or higher oligomer structure. This can be done by studying the proximity of FP and
MPER in membrane environment. Labeling of

13

C F residues in FP and

15

N F in MPER can be

done. Specific labeling of FP can be done by using labeled amino acid during peptide synthesis.
The HM can be labeled by labeling bacterially expressed protein. There is only single F residue
141

in the whole HM amino acid sequence. Then the whole ectodomain can be constructed by
native chemical ligation of labeled FP and HM. The protein construct can then be incorporated
in vesicles. REDOR experiments can be done to determine the dephasing between the 13C-15N F
residues due to proximity of the FP and MPER.

13

C-15N REDOR the distance detection limit is

approximately ~ 6-9 Å. It is expected that the distance between the NMR active nuclei would lie
between 7-9 Å if the FP and MPER are in close apposition with each other.
However, the FP and MPER might not be close enough to show adequate dephasing. Then the
next study that could be done is determination of the membrane insertion depth of MPER
included in the longer ectodomain construct. At first a qualitative estimation of membrane
insertion can be done by using 2H labeled lipid molecules with all acyl chain deuteration (D54)
and 13C F in the MPER labeled HM protein construct. Then the membrane insertion depth can
be found by determining the distance between the specific nuclei of amino acid residue in
MPER and in lipid molecule.
Another study can be done is to determine the oligomeric state of FP-HM and gp41 including
TM and cytoplasmic endodomain (Endo). Functional activity by lipid mixing assay and antibody
binding experiments can be studied by addition of TM and TM+Endo. Also analysis could be
done to determine the difference in fusion activity with the addition of TM and TM+Endo. This
could be followed by determination of proximity of TM with FP of the whole gp41 protein by
solid state NMR after being incorporated in lipid membrane environment. The distance
measurement between the FP and TM would help to understand the conformation of gp41
protein at the final stage of membrane fusion.

142

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143

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Sackett, K., Nethercott, M. J., Zheng, Z. X., and Weliky, D. P. (2014) Solid-State NMR

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Antiparallel 13 Sheet Fusion Peptide Structure in the Final Six-Helix Bundle State. Journal
of Molecular Biology 426, 1077-1094

144

APPENDICES

145

APPENDIX I
Designing of FP-HP by Native Chemical Ligation

146

Expression and Purification of Non-His Tagged Hairpin
The gp41 Hairpin construct without the His-tag was expressed in E. coli cells and purified. The
cells were lysed in glacial acetic acid and centrifuged. The supernatant contained the protein in
soluble form. The supernatant was then dialyzed (using 3500 Da MWCO dialysis tubing) against
trifluoroacetic acid: water (1:2000 v/v) in the presence of 150 μM dithiothreitol (DTT) to
prevent disulfide bond formation. After dialysis the protein solution was concentrated using a
10,000 MWCO concentrator. The concentrated protein was then purified by RP-HPLC by using
C18 column. A multistep gradient was used between 100% water / 0.1% TFA and 90%
acetonitrile / 10% water / 0.1% TFA (1). The purity of the protein was checked with SDS PAGE
(Figure AI-1) and MW was also verified by mass spectrometry. The protein concentration was
detected by measuring the absorbance at 280 nm. The total amount of protein obtained was 43
mg per 10 g of cells harvested from 1L of culture.

Figure AI-1. SDS-PAGE gel of Hairpin (non His-tag) purified by RP-HPLC. MW of Hairpin (non
His-tag) is 10.7 kDa. Lanes: 1- Hairpin; 2- Standard.
The SDS-PAGE gel of the purified Hairpin (Figure AI-1) shows sufficient purity. This protein
was used for native chemical ligation with FP.
147

Native Chemical Ligation
In order to obtain the FP-HP construct, the expressed Hairpin protein (wild type, non His-tag)
was ligated with FP. FP had labeled backbone at 13CO of L7 and at 15N amide nitrogen of F8. The
labeled FP was supplied by Dr. Kelly Sackett from our group. The labeled FP was dissolved in the
ligation buffer (8 M Guanidine chloride, 0.1 M phosphate buffer, and pH 7). The peptide was
reacted with 4-mercaptophenyl acetic acid (MPAA) catalyst to modify the thioester linked to
the C-terminus of the fusion peptide (2,3) and the mixture was incubated for 30 min. Similarly,
HP was dissolved in the ligation buffer including Tris(2-carboxyethyl)-phosphine (TCEP) (2mM)
to maintain cysteine residue in reduced state, and the two solutions were mixed together. The
reaction was allowed to proceed for 2 days in an inert (Ar) atmosphere. This was followed by
purification by RP-HPLC with a C18 column (2). Three peaks comprising of FP23, HP, and FP-HP
were collected and MW was verified by mass spectrometry. Acetonitrile was removed from the
eluted FP-HP fraction. FP-HP was allowed to refold at 4°C in the refolding buffer (20 mM
sodium formate, 200 μM TCEP, and pH-3).
The secondary structure of the whole protein was verified by CD. The signature curve of αhelical structure was observed for FP-HP as shown in the Figure AI-2. The mean residue molar
ellipticity was calculated at the minima at 222nm to be 99%.

148

CD FP-HP Refolded
Mean Residue Molar Ellipticity
(103 deg cm2 dmol-1)

10
5
0
-5
-10
-15
-20
-25
-30
-35
-40
200

210

220

230

240

250

260

Wavelength (nm)

Figure AI-2. CD spectra of the refolded FP-HP.
Purity of FP-HP was verified by SDS-PAGE (Figure AI-3). The total amount of protein obtained
was 10 mg i.e., 0.8 μmol from 4μmol of FP and 4μmol of Hairpin. This amount of protein was
sufficient for preparing a NMR (REDOR) sample. The yield of ligated FP-HP was 20% with
respect to the reactant i.e., HP.

Figure AI-3. SDS-PAGE gel comparing FP-HP and Hairpin. MW of FP-HP is 12.8 kDa, and Hairpin
(non His-tag) is 10.7 kDa. Lanes: 1 - Standard, 2 - FP-HP, 3, 4 – Hairpin.

149

APPENDIX II
Determination of Secondary Structure by Solid State NMR of Membrane
Associated FP of Gp41 Ectodomain Construct FP-HP

150

Protein Reconstitution
The synthesized FP-HP was then incorporated in membrane mimetic environment i.e., in large
unilamellar vesicles (LUVs). LUVs were prepared with ether linked lipids 1,2-di-O-tetradecyl-snglycero-3-phosphocholine (DTPC) and 1,2-di-O-tetradecyl-sn-glycero-3-phospho-rac-(1-glycerol)
sodium salt (DTPG) which lack CO nuclei. Ether linked lipids were used instead of ester linked
lipids to avoid observing the carbonyl carbon of the lipids since the chemical shift of carbonyl
carbon labeled FP-HP is desired (1,4,5). The molar proportion of lipids used was DTPC: DTPG:
Cholesterol at 8:2:5 mol ratios. The molar ratio of Lipid: Protein was 30:1. The LUVs were
prepared by dissolving the lipids in chloroform. The mixture was then dried under nitrogen
followed by vacuum overnight. Lipid dispersions were prepared by addition of 20mM sodium
formate buffer at pH-3 followed by homogenization by alternate cycles of freeze thaws.
Extrusion was done through a filter having pores of 100 nm diameter to maintain the size of the
LUVs. This was followed by addition of the protein drop wise with continuous stirring. Then it
was allowed to stand undisturbed overnight at 4°C. This was followed by centrifugation at
5000rpms for 30min and was repeated to get maximum sample out. The pellet was freeze
dried, later hydrated and then packed into a 4mm diameter magic angle spinning NMR rotor.
Solid State NMR
A REDOR of the prepared sample with backbone carbonyl carbon of L7 and amide nitrogen of
F8 of FP selectively labeled with 13C and 15N respectively was performed. The labeled FP-HP was
reconstituted in a mixture of DTPC: DTPG: Chol (8:2:5) that mimics the lipid composition of host
cells. The protein sample was cooled to -50°C to counteract heating with a rotor period of
151

125μs. 4mm magic angle spinning rotor was used and the experiment was set for 80,000
acquisitions. The pulse program used to do the REDOR experiment is explained in section 2.5.
FIDs of the S0 and S1 signal were acquired. S0 spectra contain signal of all the 13C without dipolar
coupling and S1 spectra contains signal of all the

13

C with dipolar coupling. The difference

between the FIDs of S0 and S1 gave the chemical shift of CO carbon for isotopically labeled
residue L7. The spectra S0 - S1 are shown in Figure AII-1 (right).

Figure AII-1 (Left) The structure of part of the FP showing the positions of the 13C and the 15N
labels on Leu (L7) and Phe (F8) respectively. (Right) REDOR subtracted signal for L7F8 in
DTPC:DTPG:Chol vesicles. The CO peak of L7 was referenced to the methylene peak of
adamantane at 40.5 ppm. The chemical shifts of 178.5 and 173.7 ppm correspond to α-helix
and β-sheet respectively.

152

Conclusion
Peaks at chemical shifts 178.5 and 173.7 represent α-helical and β-sheet structures respectively
for Leu residue24. The best-fit deconvoluted peaks were obtained whose integrated areas
represent the relative population of β-sheet and α-helical structure. Here it was observed that
at pH-3 the structure of FP-HP to be predominantly β-sheet (70%).

153

APPENDIX III
Study of Binding of Designed HIV Pre-Hairpin Intermediate and Membrane
Fusion Inhibitor C34 Peptide

154

Introduction
The HIV gp41 six helix bundle (SHB) structure consist of inner core formation by three Nterminal helices and the C-terminal helices bind to the conserved grooves on the surface of the
NHR trimer core (Figure AIII-1B and C) (6-8). Peptides derived from C-terminal helix region of
gp41 also known as C34 are potent HIV membrane fusion inhibitors (9,10). The efficient
inhibitory activity of C34 against various strains of HIV is probably due to high conserved
sequence of the NHR grooves to which it binds Figure AIII-1D) (6).

Figure AIII-1. (A) Schematic diagram of gp41 showing different domains – fusion peptide (fp),
NHR (Heptad repeat 1), CHR (Heptad repeat 2), transmembbane (tm) and endodomain (cyto).
The C34 peptide is shown to compare the sequence length with CHR. The three residues that
bind into the groove are shown in red. (B) Down the axis view of SHB structure of gp41
ectodomain, (C) sidewise view of SHB showing the position of CHR on the NHR core grooves. (D)
The packing of the C34 on the N36 (NHR) grooves. The N36 is represented as a molecular
surface and the three residues of C34 which fit into the binding pocket are shown in red (8).

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It has been proposed that in the process of membrane fusion of viral and host cell, at some
point the pre-hairpin intermediate (PHI) is an extended structure with the NHRs forming an
inner core coiled coil structure. It is possible that C34 which is similar to CHR helix, binds to NHR
core and stops the fusion process by inhibiting SHB formation (11). The SHB structure is very
thermally stable.
Our aim was to achieve comparatively less stable Hairpin protein with the NHR and CHR not
binding to each other and further perform structural studies. Therefore, we had designed
mutated Hairpin (HP M3) and mutated Hairpin+MPER (HM M3) as explained in Chapter 4. We
have observed that HP M3 and HM M3 are ~50% less folded than respective wild type protein
constructs. We then had hypothesized that as the secondary structure of the mutated gp41
ectodomain has the NHR and CHR interaction disturbed, then it is possible that these mutants
may bind to the C34 peptides. Based on this hypothesis, we performed some binding studies of
our protein constructs by measuring the change in the secondary structure by circular
dichroism (CD).
Experimental Conditions
The protein HM M3 was expressed in E. coli cells. The protein was purified by inclusion body
purification and IMAC. The purified protein was solubilized in 10 mM sodium formate buffer at
pH 3.0. The peptide C34 was obtained from NIH Aids Research and Reference Reagent Program
(10). The sequence is given below. Equimolar proportion of HM M3 and C34 were mixed and
allowed to incubate for some time (2 hours – overnight). After incubation, the secondary

156

structure of the mixture was measured at 20 °C and at pH 3.0. All the readings were subtracted
by buffer only CD spectra.
C34 peptide amino acid sequence (10): WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL
Results
The helicity of HM M3 is nearly 50% of the wild type HM with θ ~ -20000 deg cm2 dmol-1 (Figure
AIII-2A). The CD spectra of C34 (5 μM) only shows an unfolded structure (Figure AIII-2B). After
co-mixing of HM M3 and C34 and overnight incubation, the CD spectra (Figure AIII-3) did not
show any conspicuous difference. The protein sample was heated and then C34 was added and
allowed to incubate overnight. This additional step of heating was performed to ensure that the
NHR and CHR have least interaction between each other. However, the CD spectra still did not
show any significant change in helicity (Figure AIII-4).

157

Figure AIII-2. Circular dichroism spectra of (A) 5μM of HM M3 and (B) 5μM C34 at pH 3.0

158

Figure AIII-3. Secondary structure of HM M3 + C34, 5 μM each. The incubation was performed
overnight at 4°C. The CD was performed at pH 3.0.

Figure AIII-4. The secondary structure HM M3 + C34, 5 μM each. The protein was heated for 2
min and the C34 was added and incubated overnight at 4°C.

159

Discussion
We observed that there was no increase in the helicity of HM M3 even after the addition of C34
as hypothesized. The probable reason could be due to the unbinding of HM M3 with C34 due to
repulsive force. HM M3 has a charge of +16 and C34 has a charge of +3 at pH 3.0. As both the
protein and the peptide molecules are positively charged, it is likely that there is strong
repulsion between the two leading to unbinding.

160

APPENDIX IV
Sample Preparation of Uniformly Labeled Bacterially Expressed HIV-1 Gp41
Hairpin Ectodomain

161

The main aim of this study was to determine the oligomeric state of gp41 Hairpin (HP) protein
construct in the lipid environment. The oligomeric state of HP has been studied in aqueous
solution condition (explained in Chapter 3). We were interested to understand the oligomeric
state of the protein molecules when they are incorporated in the vesicles.
Approach
It was planned to label the HP with (1) at least 20% 13C and (2) uniformly 2H. The labeling of the
protein constructs would be optimized by running cross-polarization magic angle spinning
(MAS) solid state NMR experiments. With MAS, the polarization of abundant nuclei, i. e. 1H is
transferred to comparatively rare nuclei, in our case 13C, to achieve a very good signal to noise
ratio. This experiment would help us to understand whether the protein samples are labeled
according to our expectation. According to our hypothesis, after the labeling of the protein
samples would be complete, then the differently labeled protein samples will be incorporated
in the vesicles, followed by

13

C-2H rotational echo double resonance solid state NMR

experiments.
Materials and Method
Minimal media: For 50 mL media – (1) M9 – 0.564 g and autoclaved, (2) sterile filtered 0.1 M
CaCl2, (3) sterile filtered 1 M MgSO4. H2O, (4) Antibiotic, (5) Vitamin (MEM), (6) 100% labeled
(13C or 2H) glucose.

162

Cell Growth Conditions
The BL21 (DE3) E.coli cells were first grown in LB medium at 37 °C overnight. The cells were
then centrifuged, collected and inoculated into already prepared minimal media. After growth
for ~3 hours in minimal media, 2 mM IPTG was added for over-expression of the protein of
interest and allowed the cells to grow overnight. After the cells were harvested, lysis was done
to isolate the inclusion bodies containing labeled HP protein. Controls were done with inclusion
body pellet containing unlabeled HP protein.
NMR Experiments
Cross polarization NMR experiments were done with the help of our group member Shuang
Liang. The experiments were done at 6 kHz MAS and -50 °C. The 13CO peak of labeled protein
was referenced to the methylene peak of adamantane at 40.5 ppm.
Result
According to the cross polarization experiment, a clear

13

CO chemical shift for the 100%

13

C

labeled HP protein in inclusion body sample was observed (Figure AIV-1). Minimal signal was
observed for 20%

13

C labeled sample. There was no signal for 100% 2H labeled HP protein in

inclusion body sample. This could be due to exchange of 2H with 1H during the protein synthesis
and sample preparation steps.

163

Figure AIV-1. MAS cross polarization signal for 13C labeled inclusion body containing HP protein.
The CO peak of HP was referenced to the methylene peak of adamantane at 40.5 ppm.
Future Work
For labeling of HP protein with 2H, probably the cells have to be grown in 70-80% D2O. This
could be done by growing the cells in LB in D2O and the transferring the cells to minimal media
in D2O.

164

APPENDIX V
Location of Experimental Data Files

165

The location and file name of experimental data is listed below. There is mention of relevant
experiment/conditions at which the experiments were done.
CD
Located in Dr. Lisa Lapidus lab computer
(a) 3182014, 3192014, 3252014 – HM in DPC at pH 4.0 and 7.4
(b) Apr2013GelFiltration – HP and HM Oligomers
(c) 03112013 and 03192013– HM in 3M GuHCl
(d) 02252013 – HP after Gel Filtration
(e) 2052013 – FPHM
(f) 12112012, 12142012 – GuHCl 1-6M
(g) 12062012 – HM and HMM3 1M GuHCl
(h) 12012012, 12052012 – M3+C34
(i) 10192012 – HP and HM
(j) 5222012 – HMM2 and HMM3
(k) 02222011 – FP-HP
Fluorimeter
Located in Dr. Lisa Lapidus lab computer
POPC/POPG/Chol Experiments
(a) 2012013, 2042013, 2072013, 2082013, 02192013, 02202013, 02222013, 02232013,
02282013, 03012013, 03032013, 03042013 – HP, HM, FPHM
(b) 10262012, 10272012, 10312012, 11012012, 11022012, 11082012 – HP and HM
166

POPC/DOTAP/Chol Experiments
4142014, 4182014, Apr2_2014, Apr32_14, 4152014, Apr42014 – HP and HM
FPLC
Located in room 14 FPLC computer
(a) Koyeli/Project for Koyeli/03142013_0.3ml/min_less dilute_2CV
(b) Koyeli/Project for Koyeli/03142013_0.3ml/min_2CV
(c) Koyeli/Project for Koyeli/03142013_2.1
(d) Swetha/Method 3.8/HP(10mg_ml)DPC – 03/12/2014
NMR
(a) khafre0/mb4b/data/Koyeli_Shuang/cp_13C_ICB_061314_2
(b) khafre0/mb4b/data/Koyeli_Shuang/cp_unlabeled_P_052014
(c) khafre0/mb4b/data/Koyeli_Shuang/quecho_2H_ICB_06252014
(d) khafre0/mb4b/data/koyeli/FPH_L7F8_CPRamp_1 to 14

167

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168

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