! ! PROTEIN!AND!HYDRATION!SHELL!DYNAMICS! OF!ZNII3SUBSTITUTED!CYTOCHROME!C" By! Jennifer!Jo!Mueller! ! ! ! ! ! ! A!DISSERTATION! ! Submitted!to! Michigan!State!University! in!partial!fulfillment!of!the!requirements! for!the!degree!of! Chemistry Doctor!of!Philosophy! 2013! ! ! ! PROTEIN!AND!HYDRATION!SHELL!DYNAMICS! OF!ZNII3SUBSTITUTED!CYTOCHROME!C" By! Jennifer!Jo!Mueller! ! This!dissertation!describes!two!studies!that!provide!new!information!on!the! nature!of!the!protein!and!solvent!dynamics!that!is!probed!in!ZnIILsubstituted! cytochrome!c!(ZnCytc)!by!the!intrinsic!ZnII!porphyrin,!which!serves!as!a!probe!of! the!surrounding!protein!and!solvent.!!! In!the!first!study,!the!nature!of!the!partially!unfolded!structures!that!are! generated!in!ZnCytc!upon!optical!excitation!significantly!above!the!vibronic!origin!of! the!Q!!(S₀→S₁,!π→π*!transition)!band!was!investigated!using!continuousLwave! fluorescence!spectroscopy.!The!results!show!that!stepLlike!transitions!of!the! fluorescence!Stokes!shift!correspond!to!the!activation!threshold!for!changes!in! structure!from!the!native!state!to!a!partially!unfolded!state!associated!with!the!Ω! loop!formed!by!residues!20–35,!which!is!adjacent!to!the!Cys14!and!Cys17!thioether! linkages!from!the!porphyrin!to!the!polypeptide!backbone.!The!excitation!energy!for! optical!formation!of!the!unfolded!state!is!consistent!with!the!previous! determination!by!Englander!and!coworkers!using!hydrogenLexchange!NMR! spectroscopy!in!ferrocytochrome!c!in!the!presence!of!Gdm⁺.! In!the!second!study,!the!hydration!shell!of!ZnCytc!was!probed!using!the! indolecyanine!dye!Cy5!using!femtosecond!pump–continuumLprobe!spectroscopy.! Cy5!was!attached!to!a!surface!lysine!residue!by!a!flexible!linker!so!that!it!senses!the! ! ! viscosity!of!the!surrounding!medium!owing!to!its!nonpolar!solvation!response.!!The! main!conclusion!of!this!work!is!that!the!hydration!shell!is!as!much!as!200!times!as! viscous!as!bulk!water.!!A!simple!structural!interpretation!of!this!finding!is!that! longer!or!more!persistent!chains!of!hydrogenLbonded!water!molecules!are!present! than!in!the!bulk.! ! ! ! ! DISCLAIMER*AND*COPYRIGHT*CLAUSE* ! The!views!expressed!in!this!work!are!those!of!the!author!and!do!not!necessarily! reflect!the!official!policy!or!position!of!the!Department!of!the!Navy,!Department!of! Defense,!or!the!United!States!Government.! ! I!am!a!military!service!member.!!This!work!was!prepared!as!part!of!my!official! duties.!!Title!17,!USC,!§105!provides!that!'Copyright!protection!under!this!title!is!not! available!for!any!work!of!the!U.S.Government.'!!Title!17,!USC,!§101!defines!a!U.S.! Government!work!as!a!work!prepared!by!a!military!service!member!or!employee!of! the!U.S.!Government!as!part!of!that!person's!official!duties.! ! ! ! iv! ! TABLE*OF*CONTENTS* * LIST*OF*TABLES* *...................................................................................................................*vii* . * LIST*OF*FIGURES**..................................................................................................................*viii* . * KEY*TO*ABBREVIATIONS*..................................................................................................*xvii* * CHAPTER*1***Background*and*Significance*.....................................................................1* ! Summary! !......................................................................................................................................!1! ! 1.1!!Energy!Landscape!and!ProteinLFolding!Funnel!Theory!............................................!3! ! 1.2!!Dynamic!Solvation!.....................................................................................................................!4! ! ! 1.2.1!!Dynamic!Solvation!in!Proteins!.........................................................................!5! . ! ! 1.2.2!!Fluorescence!Stokes!Shift!Response!of!ZnCytc!..........................................!6! ! ! 1.2.3!!Intramolecular!Vibrational!Excitation!.......................................................!12! ! ! 1.2.4!!Relevance!to!Dynamics!in!LH!and!RC!Proteins!in!Photosynthesis!.!19! ! CHAPTER*2***LightJdriven*Partial*Unfolding*of*ZnIIJsubstituted*Cytochrome*c24* ! Summary! !...................................................................................................................................!24! ! 2.1!!Introduction!...............................................................................................................................!25! ! 2.2!!Experimental!Section!............................................................................................................!29! . ! ! 2.2.1!!Sample!Preparation!............................................................................................!29! ! ! 2.2.2!!ContinuousLwave!Absorption!and!Fluorescence!Spectroscopy!......!31! ! 2.3!!Results! !...................................................................................................................................!32! ! ! 2.3.1!!Dependence!of!Fluorescence!Spectra!on!Vibrational!Excitations!..!32! ! ! 2.3.2!!Temperature!Dependence!of!the!IVE!and!FR!Profiles!.........................!40! ! ! 2.3.3!!Gdm+!Dependence!of!the!IVE!Profile!..........................................................!47! ! 2.4!!Discussion!...................................................................................................................................!50! ! 2.5!!Conclusions!................................................................................................................................!54! ! CHAPTER*3***Solvation*Dynamics*of*the*Hydration*Shell*of*ZnIIJSubstituted** Cytochrome*c* *...................................................................................................................*56* . ! Summary! !...................................................................................................................................!56! ! 3.1! Introduction!.......................................................................................................................!57! ! 3.2! Experimental!Section!.....................................................................................................!61! ! ! 3.2.1! Sample!Preparation!.........................................................................................!61! . ! ! 3.2.2! Mass!Spectrometry!...........................................................................................!62! ! ! 3.2.3! ContinuousLwave!Absorption!and!Fluorescence!Spectroscopy!...!62! ! ! 3.2.4! Femtosecond!Spectroscopy!..........................................................................!63! ! ! 3.2.5! Computational!Chemistry!.............................................................................!64! . ! 3.3! Results!..................................................................................................................................!65! ! ! 3.3.1!!Mass!Spectrometry!.............................................................................................!65! ! ! 3.3.2! ContinuousLwave!Absorption!and!Fluorescence!Spectroscopy!...!65! ! v! ! ! 3.3.3!!PumpLContinuum!Probe!Spectroscopy!......................................................!69! ! 3.4! Discussion!...........................................................................................................................!82! ! CHAPTER*4***Conclusions*....................................................................................................*88* " APPENDIX* *...................................................................................................................*93* . * LITERATURE*CITED*...........................................................................................................*106* ! ! ! vi! LIST*OF*TABLES* ! ! Table!3.1! Fit!parameters!for!the!mean!frequency!model!of!Cy5!in!water! and!Cy5‑ZnCytc,!as!shown!in!Figures!3.6L3.9.!!The!parameters! correspond!to!Equation!3.1.!.................................................................................................!74! Table!3.2! Fit!parameters!for!the!720!nm!transients!modeled!to!fit!the! data!obtained!from!Cy5!in!water!and!Cy5LZnCytc,!as!shown!in!Figures! 3.10L3.13.!!The!parameters!correspond!to!Equation!3.3.!.......................................!74! ! ! ! vii! LIST*OF*FIGURES* ! Figure!1.1! Structure!of!horse!heart!ferricytochrome!c!(1HRC.pdb)! obtained!by!x‑ray!crystallography.11!Reprinted!with!permission!from! Tripathy!and!Beck,!2010!American!Chemical!Society.!The!porphyrin!is! shown!as!a!stick!figure.!The!cysteine!ligands,!Cys14!and!Cys17,!and! axial!ligands,!His18!and!Met80,!are!also!shown!as!stick!structures.!The! protein!is!otherwise!shown!as!ribbons.!The!iron!center!of!the! porphyrin!is!shown!in!magenta.!The!metal‑center!is!removed!in! fbCytc.!ZnCytc!is!obtained!by!replacing!the!iron!center!with!ZnII.9!For! interpretation!of!the!references!to!color!in!this!and!all!other!figures,! the!reader!is!referred!to!the!electronic!version!of!this!dissertation..!...................!6! Figure!1.2! Potential!energy!curves!representing!the!ground!state! (bottom)!and!excited!state!(top)!showing!the!change!in!energy!gap! between!the!two!states!following!optical!excitation.!Upon!excitation,!a! vertical!transition!occurs.!Due!to!a!small!shift!along!the!solvation! coordinate,!the!molecule!is!not!at!the!lowest!energy!state!of!the! excited‑state!potential!energy!curve.!As!the!system!moves!toward! equilibrium!in!the!excited!state,!the!energy!gap!decreases,!resulting!in! a!red!shift!of!fluorescence.!After!Maroncelli!et$al.,!1994.12!!.....................................!8! Figure!1.3! Dynamic!Stokes!shift!of!the!ZnCytc!fluorescence!spectrum.! Reprinted!with!permission!from!Lampa‑Pastirk!and!Beck,!2006,! American!Chemical!Society.!The!response!shows!an!initial!red‑shift! followed!by!a!blue‑shift!of!fluorescence!for!ZnCytc!in!water!at!room! temperature!following!excitation!in!the!Soret!band!(blue)!and!a! red‑shift!following!excitation!of!the!Q‑band!(red).!In!each!case,!the! center!frequency!of!the!0‑0!transition!was!plotted!as!a!function!of! time!delay!after!excitation.20!..............................................................................................!10! Figure!1.4! Electronic!and!vibrational!energy!levels!as!found!in!molecules! like!the!porphyrin!of!ZnCytc.!Reprinted!with!permission!from! Lampa‑Pastirk!and!Beck,!2006,!American!Chemical!Society.!After!an! initial!optical!excitation!(blue),!vibrational!relaxation!to!the!S1!state! occurs!(green),!followed!by!fluorescence!emission!(red).20!.................................!13! Figure!1.5! Absorption!(A)!and!fluorescence!(F)!dipole!strength!spectra! obtained!at!room!temperature.!Reprinted!with!permission!from! Lampa‑Pastirk!and!Beck,!2006,!American!Chemical!Society.!The! vibrational!structure!of!the!Q‑band!absorption!and!fluorescence! spectra!is!due!to!the!0‑0!and!0‑1!vibronic!transitions.20!.......................................!14! . Figure!1.6! Dependence!of!the!fluorescence!spectrum!from!ZnCytc!at!22°C! on!the!intramolecular!vibrational!excitation:!(A)!the!integrated!Stokes! ! viii! shift,!as!determined!by!the!wavenumber!of!the!0‑0!peak!maximum,! !ν 0−0 ;!(B)!ratio!of!the!dipole!strengths!of!the!0‑1!and!0‑0!peaks,! !F0−1 / F0−0 ;!and!(C)!the!half‑width!at!half‑maximum!of!the!0‑0!peak,! !Δν 0−0 .!The!plotted!abscissa!is!the!intramolecular!vibrational! excitation,!the!difference!between!the!excitation!wavenumber!and! that!of!the!0‑0!vibronic!transition.!At!the!top!of!the!figure,!the! absorption!dipole!strength!spectrum!is!plotted!with!respect!to!the! same!abscissa!scale.!The!vertical!dashed!lines!mark!the!excitation! energies!corresponding!to!apparent!activation!enthalpies!for!three! protein‑unfolding!transitions.!Reprinted!with!permission!from!Barns$ et$al.,!2008,!American!Chemical!Society.25!...................................................................!16! Figure!1.7! Time!evolution!of!the!dipole!strengths!for!the!0‑1!and!0‑0! vibronic!transitions!obtained!from!time‑resolved!fluorescence!spectra! from!ZnCytc!in!water!(22!°C).!Reprinted!with!permission!from! Lampa‑Pastirk!et$al,!2004,!American!Chemical!Society.10!Top:!Peak! intensity!normalized!by!total!dipole!strength.!Bottom:!Ratio!of!peak! intensities.!...................................................................................................................................!17! . Figure!2.1! Ribbon!(left)!and!solvent‑excluded!surface!(right)!renderings! of!the!X‑ray!crystal!structure!of!horse‑heart!ferricytochrome!c (1hrc.pdb).11!The!porphyrin!and!associated!structures!(axial!ligands:! His18!and!Met80!and!thioether!linkages:!Cys14!and!Cys17)!are!shown! as!stick!structures!in!the!ribbon!picture.!The!protein!structure!is!color! coded!from!red!to!blue!in!order!of!relative!folding!stability!following! the!scheme!of!Englander!and!coworkers:57!residues!70–85!(red),! residues!36–61!(yellow),!residues!20–35!and!the!α!helix!over!residues! 60–70!(green),!and!the!N‑!and!C‑terminal!α!helices!(blue).!Based!on!a! comparison!of!2D!NMR!spectra,!ZnCytc!is!isostructural!with!the! native,!FeII‑containing!protein!in!solution.58,59!.........................................................!26! Figure!2.2! Jablonski!energy‑level!diagram!describing!photophysical! processes!in!ZnCytc.!Vibronic!energy!levels!are!represented!by! horizontal!lines.!Absorption!of!a!photon!is!represented!by!the!vertical! blue!arrow.!Nonradiative!relaxation!(NR)!and!intersystem!crossing! (ISC)!are!indicated!by!wavy!arrows.!Ground‑state!recovery!via! fluorescence!(F)!and!phosphorescence!(P)!is!indicated!by!the!green! and!red!arrows,!respectively;!additional!nonradiative!recovery! processes!to!the!ground!state!are!not!shown.!.............................................................!28! Figure!2.3! Continuous!wave!absorption!(blue)!and!fluorescence!(red)! dipole‑strength!spectra!from!ZnCytc!at!20!°C!and!pH!7.0,! A ν /ν !and! ! F ν /ν 3 ,!respectively.!The!absorbance!and!fluorescence!spectra!were! ! normalized!to!the!amplitude!of!the!0–0!peak.!The!fluorescence! spectrum!was!excited!at!523!nm!(19120!cm−1).!The!S0→S1!vibronic! () () ! ix! transition!is!located!where!the!two!spectra!cross,!at!587.2!nm!(17030! cm−1).!!............................................................................................................................................!34! Figure!2.4! Fluorescence!dipole‑strength!spectra!from!ZnCytc!at!30!°C! with!excitation!at!(a)!523!nm!(!ν IVE = 2000 cm −1 ),!(b)!452!nm!( −1 −1 !ν IVE = 5000 cm −1 ),!(c)!425!nm!(!ν IVE = 6350 cm ),!and!(d)!408!nm!( !ν IVE = 7350 cm ).!The!same!integration!time!was!used!in!each;!the! spectra!are!plotted!with!normalization!to!the!amplitude!of!the!0–0! peak.!The!spectrum!shown!in!(a)!is!the!same!spectrum!shown!in! Figure!2.3.!....................................................................................................................................!35! Figure!2.5! Evolution!of!the!continuous‑wave!fluorescence!spectrum!from! ZnCytc!at!30!°C!as!a!function!of!!ν IVE ,!the!excitation!energy!above!the! wavenumber!of!the!S1 state!vibronic!origin:!(a)!wavenumber!of!the! peak!dipole!strength!in!the!0–0!peak!and!(b)!intensity!ratio!for!the!0– 1!and!0–0!peaks,!!F0#1 / F0#0 .!The!data!is!divided!into!four!sections! including!transitions!I,!II,!and!III,!which!were!superimposed!with! models!described!by!Equation!2.3,!and!section!IV,!which!has!a! smoothed!curve!drawn!through!the!data.!(Inset)!Parameters!for!the! sigmoidal!functions!(Equation!2.3)!used!to!describe!step!transitions!in! the!peak!wavenumber!and!fluorescence!ratio!profiles:!!x 0 ,!the!center! wavenumber!of!the!transition;!! y 0 ,!the!value!of!the!function!prior!to! the!transition;!!A ,!the!amplitude!of!the!transition;!and! σ ,!the!width!of! the!transition.!.............................................................................................................................!37! Figure!2.6! IVE!profiles!for!ZnCytc!as!a!function!of!temperature.!Curves!at! each!temperature!were!obtained!by!fitting!the!experimental!peak! wavenumbers!(Equation!2.3).!The!integrated!Stokes!shift,!as! determined!by!the!wavenumber!of!the!0–0!peak!maximum,!!ν 0#0 .!Peak! maxima!are!plotted!as!points.!The!trendline!includes!a!fit!using! Equation!2.3!to!describe!each!of!the!first!three!apparent!transitions!in! the!data!and!a!smoothed!curve!to!describe!the!data!after!the!peak.! (a)!The!integrated!Stokes!shift!at!eight!temperatures!(5!°C,!10!°C,! 20!°C,!30!°C,!40!°C,!50!°C,!60!°C,!and!70!°C)!demonstrating!the!change! in!the!profile!as!temperature!increases!to!the!denaturation!point!of! the!protein.!(b)!The!integrated!Stokes!shift!at!four!temperatures! (70!°C,!80!°C,!85!°C,!and!90!°C),!demonstrating!the!change!in!the! profile!as!temperature!increases!above!the!denaturation!point!of!the! protein.!..........................................................................................................................................!41! Figure!2.7! Dependence!of!the!fluorescence!peak!ratio!on!intramolecular! vibrational!excitation!at!multiple!temperatures.!A!line!graph!for!each! temperature!shows!the!actual!data!points!obtained!after!smoothing! the!fluorescence!curve!and!using!a!peak‑finding!routine!to!identify!the! peak!position!and!intensity.!.................................................................................................!42! ! x! Figure!2.8! !Temperature!dependences!of!the!midpoints,! x 0,i ,!for!the!IVE! ! and!FR!profiles!from!ZnCytc:!(a)!Transition!I;!(b)!Transition!II;!(c)! Transition!III.!For!(a)!and!(b),!the!trendlines!shown!at!low! temperatures!includes!data!points!ranging!from!5°C!to!50°C;!the! trendlines!shown!at!higher!temperatures!includes!data!points!from! 70°C!to!90°C.!For!(c),!the!trendline!shown!at!low!temperatures! includes!data!points!ranging!from!5°C!to!30°C;!the!trendline!at!higher! temperatures!includes!data!points!from!30°C!to!90°C.!...........................................!45! Figure!2.9! Absolute!value!of!the!amplitudes!of!the!first!red!shift,!!A 1 ,!and! the!blue!shift,!!A 2+3 ,!of!the!IVE!profile!as!a!function!of!temperature.! For!(a)!and!(b),!the!trendlines!shown!at!low!temperatures!includes! data!points!ranging!from!5°C!to!70°C!(slope:!0.003!cm–1/°C!and!0.054! cm–1/°C,!respectively),!below!the!protein’s!transition!to!a!denatured! state.!The!trendlines!shown!at!higher!temperatures!includes!data! points!from!70°C!to!90°C!(slope:!1.03!cm–1/°C!and!2.18!cm–1/°C,! respectively),!after!the!protein!is!denatured.!..............................................................!46! . Figure!2.10! Dependence!of!the!IVE!profile!of!ZnCytc!as!a!function!of!!ν IVE ! on!Gdm+!concentration.!The!integrated!Stokes!shift!at!seven! concentrations!of!guanidinium!ion!(0.0!M,!1.0!M,!1.5!M,!2.0!M,!2.5!M,! 3.0!M,!and!4.0!M),!demonstrating!the!change!in!the!profile!as! denaturant!concentration!increases!until!the!protein!is!fully! denatured.!....................................................................................................................................!47! Figure!2.11! The!excess!excitation!wavenumber!midpoint,!!x 0 ,!of!each! transition!as!a!function!of!guanidinium!concentration,!(a)!!x 0,I ,!(slope:! 201!cm–1/M)!(b)! x 0,II ,!(slope:!137!cm–1/M)!(c)! x 0,III ,! ! ! ‑ (slope:!34!cm 1/M).!.................................................................................................................!49! Figure!2.12! Absolute!value!of!the!amplitude!of!the!red!shift!of!the!ZnCytc! fluorescence!spectrum!in!transition!I,!!A 1 ,!and!the!sum!of!the! amplitudes!for!the!blue!shift,!!A2 + A3 ,!in!the!IVE!profile!as!a!function! of!guanidinium!ion!concentration.!For!both!(a)!and!(b),!the!trendlines! shown!at!low!Gdm+!concentration!include!data!points!ranging!from! 0.0!M!to!1.0!M!(slope:!2.3!cm–1/°C!and!18.3!cm–1/°C,!respectively),! below!the!protein’s!transition!to!a!denatured!state.!The!trendlines! shown!at!higher!Gdm+!concentration!includes!data!points!from!2.5!M! to!5.0!M!(slope:!–4.5!cm–1/°C!and!–9.1!cm–1/°C,!respectively),!after! the!protein!is!denatured.!.......................................................................................................!50! Figure!2.13! Another!view!of!the!X‑ray!crystal!structure!of!horse‑heart! ferricytochrome!c (1hrc.pdb)11!following!the!scheme!of!Englander! and!coworkers.57!The!figure!has!been!rotated!when!compared!to! Figure!2.1!to!allow!a!view!of!the!two!thioether!linkages,!Cys14!and! ! xi! Cys17.!Also!note!the!blue!dot!representing!an!intrinsic!water!molecule! adjacent!to!the!porphyrin.!....................................................................................................!52! Figure!3.1! Structures!of!(a)!Cy5–lysine!adduct;!(b)!B3LYP/6‑31G(d)! structure!for!the!ground‑state!Cy5!chromophore;!(c)!horse‑heart! ferricytochrome!c (1HRC.pdb).11!The!protein!is!shown!in!a!ribbon! representation;!the!heme,!amino!acid!residues!Met80,!His18,!Cys14,! Cys17,!and!the!side!chains!of!the!lysine!residues!are!shown!in!a!stick! representation.!The!polypeptide!is!color!coded!from!red!to!blue! following!the!scheme!of!Englander!and!coworkers:57!residues!70–85! (red),!residues!36–61!(yellow),!residues!20–35!and!60–70!(green),! and!the!N‑!and!C‑terminal!α!helices!(blue).!..................................................................!59! Figure!3.2! Continuous‑wave!absorption!(blue)!and!fluorescence!(red)! spectra!of!Cy5!in!water!overlaid!with!the!laser!spectrum!(black)!and! normalized!to!maximum!peak!intensity.!........................................................................!67! Figure!3.3! Continuous‑wave!absorption!(blue)!and!fluorescence!(red)! spectra!of!Cy5–ZnCytc!overlaid!with!the!pump!spectrum!(black)!and! normalized!to!maximum!peak!intensity.!........................................................................!67! Figure!3.4! Time‑resolved!pump‑probe!spectra!for!Cy5!in!water!with! probe!delays!from!−20!fs!to!54!ps.!....................................................................................!71! Figure!3.5! Time‑resolved!pump‑probe!spectra!for!Cy5–ZnCytc!with!probe! delays!from!−20!fs!to!58!ps.!.................................................................................................!72! Figure!3.6! Time!evolution!of!the!mean!frequency!of!the!pump‑probe! spectrum!of!Cy5!in!water!at!short!delay!times.!The!data!points!are! overlaid!with!a!fit!to!Equation!3.1,!and!the!fit!parameters!are!listed!in! Table.!3.1.!.....................................................................................................................................!75! Figure!3.7! Time!evolution!of!the!mean!frequency!of!the!pump‑probe! spectrum!of!Cy5!in!water.!The!data!points!are!overlaid!with!a!fit!to! Equation!3.1,!and!the!fit!parameters!are!listed!in!Table.!3.1.!................................!75! Figure!3.8! Time!evolution!of!the!mean!frequency!of!the!pump‑probe! spectrum!of!Cy5–ZnCytc!at!short!delay!times.!The!data!points!are! overlaid!with!a!fit!to!Equation!3.1,!and!the!fit!parameters!are!listed!in! Table.!3.1.!.....................................................................................................................................!76! Figure!3.9! Time!evolution!of!the!mean!frequency!of!the!pump‑probe! spectrum!of!Cy5–ZnCytc.!The!data!points!are!overlaid!with!a!fit!to! Equation!3.1,!and!the!fit!parameters!are!listed!in!Table.!3.1.!................................!76! Figure!3.10! Pump‑probe!transient!of!Cy5!in!water!with!the!probe! wavelength!at!720!nm.!The!data!points!are!overlaid!with!a!fit!to! Equation!3.3;!the!fit!parameters!are!listed!in!Table!3.2.!The!delay! ! xii! spacing!in!this!experiment!shows!vibrational!coherence!not!observed! with!greater!step‑size.!............................................................................................................!78! Figure!3.11! Pump‑probe!transient!of!Cy5!in!water!with!the!probe! wavelength!at!720!nm.!The!data!points!are!overlaid!with!a!fit!to! Equation!3.3;!the!fit!parameters!are!listed!in!Table!3.2.!.........................................!78! Figure!3.12! Pump‑probe!transient!of!Cy5–ZnCytc!with!the!probe! wavelength!at!720!nm.!The!data!points!are!overlaid!with!a!fit!to! Equation!3.3;!the!fit!parameters!are!listed!in!Table!3.2.!The!delay! spacing!in!this!experiment!shows!vibrational!coherence!not!observed! with!greater!step‑size.!............................................................................................................!79! Figure!3.13! Pump‑probe!transient!of!Cy5–ZnCytc!with!the!probe! wavelength!at!720!nm.!The!data!points!are!overlaid!with!a!fit!to! Equation!3.3;!the!fit!parameters!are!listed!in!Table!3.2.!.........................................!79! Figure!3.14! Oscillatory!residuals!(data!−!fit)!from!the!pump‑probe! transient!of!Cy5!in!water!with!the!probe!wavelength!at!720!nm.!The! data!points!are!fit!to!a!set!of!damped!cosinusoids!using!a!LP‑SVD! program.!The!spectral!density!obtained!from!the!fit!is!shown!in! Figure!3.16.!..................................................................................................................................!80! Figure!3.15! Oscillatory!residuals!(data!−!fit)!from!the!pump‑probe! transient!of!Cy5–ZnCytc!with!the!probe!wavelength!at!720!nm.!The! data!points!are!fit!to!a!set!of!damped!cosinusoids!using!a!LP‑SVD! program.!The!spectral!density!obtained!from!the!fit!is!shown!in! Figure!3.17.!..................................................................................................................................!80! Figure!3.16! Spectral!density!obtained!from!the!LP‑SVD!fit!of!the!oscillatory! part!of!the!pump‑probe!transient!of!Cy5!in!water!with!the!probe! wavelength!at!720!nm!(see!Figure!3.14).!.......................................................................!81! Figure!3.17! Spectral!density!obtained!from!the!LP‑SVD!fit!of!the!oscillatory! part!of!the!pump‑probe!transient!of!Cy5–ZnCytc!with!the!probe! wavelength!at!720!nm!(see!Figure!3.15).!Three!frequency!components! at!286!cm−1,!420!cm−1,!and!538!cm−1!were!found!for!Cy5–ZnCytc.!.................!82! Figure!A1! IVE!profile!of!ZnCytc!at!5!°C:!Evolution!of!the!wavenumber!of! the!peak!dipole!strength!in!the!0–0!peak!of!the!continuous‑wave! fluorescence!spectrum!from!ZnCytc!at!5!°C!as!a!function!of!!ν IVE ,!the! excitation!energy!above!the!wavenumber!of!the!0–0!absorption!peak.!..........!95! . Figure!A2! IVE!profile!of!ZnCytc!at!10!°C:!Evolution!of!the!wavenumber!of! the!peak!dipole!strength!in!the!0–0!peak!of!the!continuous‑wave! fluorescence!spectrum!from!ZnCytc!at!10!°C!as!a!function!of!!ν IVE ,!the! excitation!energy!above!the!wavenumber!of!the!0–0!absorption!peak.!..........!95! . ! xiii! Figure!A3! IVE!profile!of!ZnCytc!at!20!°C:!Evolution!of!the!wavenumber!of! the!peak!dipole!strength!in!the!0–0!peak!of!the!continuous‑wave! fluorescence!spectrum!from!ZnCytc!at!20!°C!as!a!function!of!!ν IVE ,!the! excitation!energy!above!the!wavenumber!of!the!0–0!absorption!peak.!..........!96! . Figure!A4! IVE!profile!of!ZnCytc!at!30!°C:!Evolution!of!the!wavenumber!of! the!peak!dipole!strength!in!the!0–0!peak!of!the!continuous‑wave! fluorescence!spectrum!from!ZnCytc!at!30!°C!as!a!function!of!!ν IVE ,!the! excitation!energy!above!the!wavenumber!of!the!0–0!absorption!peak.!..........!96! . Figure!A5! IVE!profile!of!ZnCytc!at!40!°C:!Evolution!of!the!wavenumber!of! the!peak!dipole!strength!in!the!0–0!peak!of!the!continuous‑wave! fluorescence!spectrum!from!ZnCytc!at!40!°C!as!a!function!of!!ν IVE ,!the! excitation!energy!above!the!wavenumber!of!the!0–0!absorption!peak.!..........!97! . Figure!A6! IVE!profile!of!ZnCytc!at!50!°C:!Evolution!of!the!wavenumber!of! the!peak!dipole!strength!in!the!0–0!peak!of!the!continuous‑wave! fluorescence!spectrum!from!ZnCytc!at!50!°C!as!a!function!of!!ν IVE ,!the! excitation!energy!above!the!wavenumber!of!the!0–0!absorption!peak.!..........!97! . Figure!A7! IVE!profile!of!ZnCytc!at!60!°C:!Evolution!of!the!wavenumber!of! the!peak!dipole!strength!in!the!0–0!peak!of!the!continuous‑wave! fluorescence!spectrum!from!ZnCytc!at!60!°C!as!a!function!of!!ν IVE ,!the! excitation!energy!above!the!wavenumber!of!the!0–0!absorption!peak.!..........!98! . Figure!A8! IVE!profile!of!ZnCytc!at!70!°C:!Evolution!of!the!wavenumber!of! the!peak!dipole!strength!in!the!0–0!peak!of!the!continuous‑wave! fluorescence!spectrum!from!ZnCytc!at!70!°C!as!a!function!of!!ν IVE ,!the! excitation!energy!above!the!wavenumber!of!the!0–0!absorption!peak.!..........!98! . Figure!A9! IVE!profile!of!ZnCytc!at!80!°C:!Evolution!of!the!wavenumber!of! the!peak!dipole!strength!in!the!0–0!peak!of!the!continuous‑wave! fluorescence!spectrum!from!ZnCytc!at!80!°C!as!a!function!of!!ν IVE ,!the! excitation!energy!above!the!wavenumber!of!the!0–0!absorption!peak.!..........!99! . Figure!A10! IVE!profile!of!ZnCytc!at!85!°C:!Evolution!of!the!wavenumber!of! the!peak!dipole!strength!in!the!0–0!peak!of!the!continuous‑wave! fluorescence!spectrum!from!ZnCytc!at!85!°C!as!a!function!of!!ν IVE ,!the! excitation!energy!above!the!wavenumber!of!the!0–0!absorption!peak.!..........!99! . Figure!A11! IVE!profile!of!ZnCytc!at!90!°C:!Evolution!of!the!wavenumber!of! the!peak!dipole!strength!in!the!0–0!peak!of!the!continuous‑wave! fluorescence!spectrum!from!ZnCytc!at!90!°C!as!a!function!of!!ν IVE ,!the! excitation!energy!above!the!wavenumber!of!the!0–0!absorption!peak.!........! 00! . 1 Figure!A12! IVE!profile!of!ZnCytc!in!the!presence!of!0.0!M!Gdm+:!Evolution! of!the!wavenumber!of!the!peak!dipole!strength!in!the!0–0!peak!of!the! continuous‑wave!fluorescence!spectrum!from!ZnCytc!at!20!°C!as!a! ! xiv! function!of!!ν IVE ,!the!excitation!energy!above!the!wavenumber!of!the! 0–0!absorption!peak.!............................................................................................................! 00! . 1 Figure!A13! IVE!profile!of!ZnCytc!in!the!presence!of!0.25!M!Gdm+:! Evolution!of!the!wavenumber!of!the!peak!dipole!strength!in!the!0–0! peak!of!the!continuous‑wave!fluorescence!spectrum!from!ZnCytc!at! 20!°C!as!a!function!of!!ν IVE ,!the!excitation!energy!above!the! wavenumber!of!the!0–0!absorption!peak.!...................................................................! 01! 1 Figure!A14! IVE!profile!of!ZnCytc!in!the!presence!of!0.5!M!Gdm+:!Evolution! of!the!wavenumber!of!the!peak!dipole!strength!in!the!0–0!peak!of!the! continuous‑wave!fluorescence!spectrum!from!ZnCytc!at!20!°C!as!a! function!of!!ν IVE ,!the!excitation!energy!above!the!wavenumber!of!the! 0–0!absorption!peak.!............................................................................................................! 01! . 1 Figure!A15! IVE!profile!of!ZnCytc!in!the!presence!of!1.0!M!Gdm+:!Evolution! of!the!wavenumber!of!the!peak!dipole!strength!in!the!0–0!peak!of!the! continuous‑wave!fluorescence!spectrum!from!ZnCytc!at!20!°C!as!a! function!of!!ν IVE ,!the!excitation!energy!above!the!wavenumber!of!the! 0–0!absorption!peak.!............................................................................................................! 02! . 1 Figure!A16! IVE!profile!of!ZnCytc!in!the!presence!of!1.5!M!Gdm+:!Evolution! of!the!wavenumber!of!the!peak!dipole!strength!in!the!0–0!peak!of!the! continuous‑wave!fluorescence!spectrum!from!ZnCytc!at!20!°C!as!a! function!of!!ν IVE ,!the!excitation!energy!above!the!wavenumber!of!the! 0–0!absorption!peak.!............................................................................................................! 02! . 1 Figure!A17! IVE!profile!of!ZnCytc!in!the!presence!of!2.0!M!Gdm+:!Evolution! of!the!wavenumber!of!the!peak!dipole!strength!in!the!0–0!peak!of!the! continuous‑wave!fluorescence!spectrum!from!ZnCytc!at!20!°C!as!a! function!of!!ν IVE ,!the!excitation!energy!above!the!wavenumber!of!the! 0–0!absorption!peak.!............................................................................................................! 03! . 1 Figure!A18! IVE!profile!of!ZnCytc!in!the!presence!of!2.5!M!Gdm+:!Evolution! of!the!wavenumber!of!the!peak!dipole!strength!in!the!0–0!peak!of!the! continuous‑wave!fluorescence!spectrum!from!ZnCytc!at!20!°C!as!a! function!of!!ν IVE ,!the!excitation!energy!above!the!wavenumber!of!the! 0–0!absorption!peak.!............................................................................................................! 03! . 1 Figure!A19! IVE!profile!of!ZnCytc!in!the!presence!of!3.0!M!Gdm+:!Evolution! of!the!wavenumber!of!the!peak!dipole!strength!in!the!0–0!peak!of!the! continuous‑wave!fluorescence!spectrum!from!ZnCytc!at!20!°C!as!a! function!of!!ν IVE ,!the!excitation!energy!above!the!wavenumber!of!the! 0–0!absorption!peak.!............................................................................................................! 04! . 1 Figure!A20! IVE!profile!of!ZnCytc!in!the!presence!of!4.0!M!Gdm+:!Evolution! of!the!wavenumber!of!the!peak!dipole!strength!in!the!0–0!peak!of!the! continuous‑wave!fluorescence!spectrum!from!ZnCytc!at!20!°C!as!a! ! xv! function!of!!ν IVE ,!the!excitation!energy!above!the!wavenumber!of!the! 0–0!absorption!peak.!............................................................................................................! 04! . 1 Figure!A21! IVE!profile!of!ZnCytc!in!the!presence!of!5.0!M!Gdm+:!Evolution! of!the!wavenumber!of!the!peak!dipole!strength!in!the!0–0!peak!of!the! continuous‑wave!fluorescence!spectrum!from!ZnCytc!at!20!°C!as!a! function!of!!ν IVE ,!the!excitation!energy!above!the!wavenumber!of!the! 0–0!absorption!peak.!............................................................................................................! 05! . 1 ! ! ! xvi! KEY*TO*ABBREVIATIONS* ! 3PEPS,!threeLpulse!photon!echo!peak!shift! Chl,!chlorophyll! ESA,!excited!state!absorption! fbCytc,!freeLbase!cytochrome!c!(metalLfree)! FeCytc,!ferric!cytochrome!c$ FRET,!Förster!resonance!energy!transfer! FSS,!fluorescence!Stokes!shift! fwhm,!fullLwidth!at!half!maximum! Gdm+,!guanidinium!ion! HXLNMR,!hydrogenLexchange!nuclear!magnetic!resonance!imaging! IVE,!intramolecular!vibrational!excitation! IVR,!intramolecular!vibrational!redistribution! LH,!lightLharvesting! LHCII,!lightLharvesting!complex!II! LPLSVD,!linear!predictionLsingular!value!decomposition! Lut,!lutein! MALDILTOF!MS,!matrixLassisted!desorption/ionization!timeLofLflight!mass! spectrometry! Neo,!neoxanthin! NHS,!NLhydroxysuccinimidyl! NPQ,!nonLphotochemical!quenching! ! xvii! OPA,!optical!parametric!amplifier! PB,!photobleaching! Vio,!violaxanthin! RC,!reaction!center! SE,!stimulated!emission! ZnCytc,!zincIILsubstituted!cytochrome!c!!! ! ! xviii! ! CHAPTER!1! ! Background!and!Significance! Summary! This!dissertation!presents!two!studies!of!protein!and!solvent!dynamics!in!a!model! for!a!single‑chlorophyll‑binding!chromoprotein:!ZnII‑substituted!cytochrome!c! (ZnCytc.)!The!main!goals!of!this!work!were!to!determine!how!the!structure!of!the! protein!dissipates!excess!vibrational!energy!and!to!determine!how!the!surrounding! hydration!shell!is!coupled!to!and!damps!these!protein!motions.!This!is!particularly! relevant!to!photosynthetic!systems,!which!use!chromophores!to!absorb!a!broad! solar!spectrum!and!harmlessly!dissipate!unneeded!energy.!The!single!ZnII! porphyrin!in!ZnCytc!is!an!analog!to!the!photosynthetic!chromophores!and!acts!as!an! intrinsic!probe!in!these!studies.!An!additional!point!of!significance!that!is!distinct! from!photosynthesis!comes!from!the!possibility!that!the!vibrational!excitation!can! be!used!to!generate!partially!unfolded!structures.!The!proteins!could!then!be! studied!as!they!refold!spontaneously.!! In!this!first!chapter,!we!review!the!background!for!these!two!areas!with!an! emphasis!on!the!theory!of!protein!folding,!previous!studies!and!the!background!that! led!to!this!work,!and!a!brief!review!of!photosynthesis.!The!goal!of!the!first!part!of! this!work!was!to!test!hypotheses!proposed!in!previous!research!on!intramolecular! vibrational!excitation!(IVE).!IVE!was!proposed!as!a!method!to!study!protein! unfolding!and!refolding!as!an!alternative!to!current!methods!which!rely!on!chemical! ! 1! and!thermal!denaturation.!The!significance!of!this!question!relates!to!using!a!new! method!to!reproducibly!denature!a!protein!under!solution!conditions!favoring!the! native!state!using!vibration!obtained!via!optical!excitation!and!to!provide!further! insight!into!transfer!of!vibrational!energy!in!photosynthetic!systems.!The!goal!of!the! second!part!of!this!work!was!to!study!differences!in!the!behavior!of!water!molecules! in!the!hydration!shell!of!a!protein!compared!to!water!molecules!in!bulk!water.!The! results!indicate!that!water!molecules!in!the!hydration!shell!of!ZnCytc!exhibit!higher! viscosity!than!water!molecules!in!the!bulk,!which!hinder!motion!of!other!molecules! in!that!region.!These!findings!improve!our!understanding!of!the!role!played!by!the! hydration!layer!in!stabilizing!the!native!fold!and!damping!its!unfolding!motions.! ! ! ! 2! 1.1!Energy!Landscape!and!Protein‑Folding!Funnel!Theory! Proteins!in!solution!tend!to!exhibit!two!states,!the!native!state!and!the!denatured! state,!but!a!microscopic!analysis!of!protein!crystal!structure!suggests!a!number!of! alternate!conformations.!The!energy!landscape/protein‑folding!funnel!picture!is!an! attempt!to!rationalize!this!paradox.!The!energy!landscape!picture!introduced!by! Frauenfelder,!Sligar,!and!Wolynes,1!is!a!way!to!describe!the!potential!energy!of!a! protein!as!a!function!of!protein!conformations.!The!potential‑energy!surface!is! envisioned!as!a!surface!modulated!by!very!large!number!of!minima,!which!define! the!different!conformations!that!the!polypeptide!can!adopt.!The!barriers!between! these!minima!sort!the!landscape!into!tiers!according!to!their!heights!compared!to! thermal!fluctuations!(!ΔE = kBT ).!Under!specific!solution!conditions!and!at!a!given! temperature,!there!are!many!possible!conformations!for!a!protein.!The!full!energy! landscape!encompasses!all!conformational!states!the!protein!can!access!given!a!full! range!of!solution!conditions!and!temperatures.!Under!solution!conditions!favoring! the!native!state,!a!protein!is!free!to!access!only!the!part!of!the!energy!landscape!with! low!energy!barriers!between!potential!wells.!In!order!to!reach!another!tier!on!the! hierarchy!of!conformational!states,!more!activation!energy!is!required.!The!native! state!can!be!disrupted!by!adding!energy!in!the!form!of!heat,!which!can!overcome!the! activation!barriers,!or!by!adding!a!denaturant,!which!can!alter!the!shape!of!the! activation!barriers!and!allow!access!to!previously!inaccessible!conformations.! Onuchic!and!Wolynes2!suggested!that!the!energy!landscape!for!a!biologically! active!polypeptide!is!a!funnel‑shaped!landscape!that!leads!an!unfolded!protein! toward!the!native!state!as!it!seeks!lower!energy!configurations,!with!the!native!state! ! 3! at!the!minimum!of!the!potential!energy!surface.!Under!solution!conditions!that!favor! the!native!state,!the!protein!is!effectively!trapped!at!the!bottom!of!the!potential! energy!well.!Adding!heat!to!the!system!by!increasing!the!temperature!will!allow!the! protein!to!sample!more!conformations,!effectively!climbing!the!sides!of!the!funnel.! Alternatively,!adding!a!denaturant!to!the!system!alters!the!shape!of!the!energy! landscape,!which!may!also!allow!the!protein!to!sample!a!wider!variety!of! conformations!than!are!accessible!under!native‑state!conditions.!It!is!expected!that! there!are!a!variety!of!paths!along!the!energy!landscape/protein‑folding!funnel!for!a! protein!to!follow!as!it!approaches!the!native!state!rather!than!a!single!sequential! pathway!following!specific!intermediate!states.3R5!This!leads!to!the!questions!of! how!protein!intermediates!move!toward!the!native!state,!whether!these!pathways! can!be!observed,!and!how!the!hydration!shell!of!the!protein!is!involved!in!directing! motion!over!activation!barriers!along!the!energy!landscape.! 1.2!Dynamic!Solvation! The!study!of!dynamic!solvation!investigates!how!solvent!molecules!move!or!are! reorganized!in!response!to!a!change!in!chemical!or!electronic!structure!of!a!solute.! In!liquids,!solvent!dynamics!have!frequently!been!studied!using!time‑resolved! fluorescence!spectroscopy.!After!optical!excitation,!the!fluorescence!spectrum!shifts! to!the!red!because!the!solvent!molecules!reorganize!around!the!fluorescent!probe! solute,!which!typically!has!a!larger!dipole!moment!in!the!excited!state.!Nonpolar! solvation!involves!reorganization!due!to!changes!in!the!probe's!structure!or!size!as!a! result!of!the!optical!excitation.!! ! 4! 1.2.1!Dynamic!Solvation!in!Proteins! The!fluctuations!of!proteins!due!to!diffusive!motion!on!the!energy!landscape! from!minimum!to!minimum!can!be!characterized!using!a!similar!approach!to!that! used!to!characterize!dynamic!solvation!in!liquids.!The!first!work!in!this!area!was! performed!by!McLendon,!Mukamel,!and!coworkers,6!who!characterized!the! fluorescence!Stokes'!shift!(FSS)!response!of!apomyoglobin!with!an!extrinsic! chromophore.!Replacement!of!the!native!heme!with!a!fluorescent!porphyrin!makes! the!myoglobin!suitable!for!picosecond!spectroscopy!experiments.!Similar! experiments!were!performed!by!the!Beck!group!on!the!femtosecond!timescale!in!in! the!alpha!subunit!of!C‑phycocyanin!and!in!ZnCytc.7!Equilibrium!fluctuations!can!be! measured!in!proteins!by!dynamic!solvation!because!proteins!exhibit!some! properties!of!liquids.8!By!treating!a!protein!in!solution!as!a!liquid,!responses!can!be! studied!using!dynamic!Stokes!shift!experiments7,9,10!Using!time‑resolved! fluorescence!spectroscopy,!Beck!and!Tripathy!showed!that!the!activation!enthalpy! for!unfolding!of!free‑base!cytochrome!c!(fbCytc)!is!significantly!lower!than!for! ZnCytc!(structure!of!cytochrome!c!shown!in!Figure!1.1)!and!that!the!time!scales!of! dynamic!solvation!change!due!to!structural!perturbation.!They!also!showed!that!the! fluorescence!Stokes!shift!(FSS)!response!function!of!fbCytc!is!significantly!slower! than!that!of!ZnCytc,!due!to!an!increase!of!core!and!surface!fluctuations!relative!to! ZnCytc.9! ! 5! ! Figure!1.1.!Structure!of!horse!heart!ferricytochrome!c!(1HRC.pdb)!obtained!by! x‑ray!crystallography.11!Reprinted!with!permission!from!Tripathy!and!Beck,!2010! American!Chemical!Society.!The!porphyrin!is!shown!as!a!stick!figure.!The!cysteine! ligands,!Cys14!and!Cys17,!and!axial!ligands,!His18!and!Met80,!are!also!shown!as! stick!structures.!The!protein!is!otherwise!shown!as!ribbons.!The!iron!center!of!the! porphyrin!is!shown!in!magenta.!The!metal‑center!is!removed!in!fbCytc.!ZnCytc!is! obtained!by!replacing!the!iron!center!with!ZnII.9!For!interpretation!of!the!references! to!color!in!this!and!all!other!figures,!the!reader!is!referred!to!the!electronic!version! of!this!dissertation.! 1.2.2!Fluorescence!Stokes!Shift!Response!of!ZnCytc! Tripathy!and!Beck!reviewed!the!theory!and!application!of!the!FSS!response!in! their!studies!of!dynamic!solvation!in!liquids!and!in!proteins.9!The!potential!energy! curve!of!the!excited!state!of!the!intrinsic!chromophore!was!thought!to!be!shifted! horizontally!along!the!solvation!coordinate!relative!to!the!potential!energy!curve!of! the!ground!state!(see!Figure!1.2).!The!expected!shift!meant!that!the!excited!molecule! was!not!at!the!equilibrium!position!on!its!potential!energy!curve,!so!forces!on!the! ! 6! molecule!pushed!it!toward!equilibrium.!Subsequently,!when!the!molecule!relaxed! by!emitting!radiatively,!the!energy!gap!between!the!ground!and!excited!states!was! smaller!than!the!gap!of!the!vertical!transition!from!equilibrium!position!of!the! ground!state!to!the!excited!state.!Thus,!the!time‑resolved!fluorescence!spectrum! shifts!to!the!red!as!time!increases!and!eventually!reaches!an!equilibrium!value.! These!shifts!were!measured!from!a!set!of!picosecond!time‑resolved!fluorescence! spectra,!which!were!obtained!as!slices!of!a!time–intensity–wavelength!surface! assembled!from!a!series!of!single‑wavelength!fluorescence!transients!acquired!with! the!detection!wavelength!scanned!across!the!fluorescence!spectrum.!Time‑resolved! spectra!were!obtained!by!assembling!the!data!as!a!time‑wavenumber‑intensity! surface!and!slicing!the!surface!at!specific!delay!times.! ! 7! ! Figure!1.2.!Potential!energy!curves!representing!the!ground!state!(bottom)!and! excited!state!(top)!showing!the!change!in!energy!gap!between!the!two!states! following!optical!excitation.!Upon!excitation,!a!vertical!transition!occurs.!Due!to!a! small!shift!along!the!solvation!coordinate,!the!molecule!is!not!at!the!lowest!energy! state!of!the!excited‑state!potential!energy!curve.!As!the!system!moves!toward! equilibrium!in!the!excited!state,!the!energy!gap!decreases,!resulting!in!a!red!shift!of! fluorescence.!After!Maroncelli!et$al.,!1994.12!! () The!FSS!is!also!called!the!solvent‑response!function,! Sν t 13R16!It!describes! ! reorganization!of!solvent!molecules!surrounding!a!molecule!in!a!condensed!phase! medium!that!occurs!after!electronic!excitation.!An!electronic!excitation!from!the! ! 8! ground!state!to!the!first!excited!state,!S1,!is!generally!accompanied!by!a!change!in! () the!dipole!moment!of!the!molecule,!the!probe!chromophore.9! Sν t !has!been! ! defined!using!the!time!evolution!of!the!mean!frequency!of!the!time‑resolved! fluorescence!spectrum:! ! ! ( ) (( )) (( )) ν t −ν ∞ ! Sν t = ν 0 −ν ∞ ! (1.1)! () In!polar!liquids,!most!properties!of! Sν t !observed!experimentally!have!been! ! calculated!by!treating!the!solvent!as!a!dielectric!continuum.13,17!The!Beck!Lab!has! treated!the!optical!transition!of!the!probe!as!a!step‑function!change!in!the!electric! field!to!which!surrounding!solvent!molecules!react.9!There!are!fluctuations!of!the! local!electric!field!due!to!random!motions!of!solvent!molecules!and!the!change!in! orientation!of!their!dipole!moment!around!the!chromophore.!These!fluctuations! () help!to!understand!the!molecular!character!of! Sν t .9!In!the!time!domain,!the! ! ground‑to‑excited!state!transition!frequency,!!ω = 2πν ,!of!the!probe!exhibits! fluctuations!from!the!frequency!averaged!over!time!or!the!frequency!averaged! instantaneously!over!the!ensemble.!Those!fluctuations!are!described!by!a! () time‑correlation!function!for!the!probe's!optical!transition,! M t ,! ! Δω 0 Δω t ! ! M t = 2 ! Δω ! () () () ( ) (1.2)! () that!describes!the!loss!of!memory!of!the!system!over!time.! M t !is!equivalent!to!the! ! fluorescence!Stokes'!shift!in!the!linear!response!regime!where!the! () fluctuation‑dissipation!relation!holds.! M t !is!determined!from!the!time!averaged! ! () and!instantaneous!ground‑to‑excited!state!transition!frequencies,! ω !and! ω t ,! ! ! 9! () () respectively,!and! Δω t = ω − ω t ,!the!fluctuation!averaged!over!the!ensemble.13,14! ! At!reference!time,!t=0,!there!is!no!loss!of!memory!of!the!instantaneous!transition! () frequency,! ω 0 ,!but!as!time!evolves,!the!system!retains!less!and!less!of!its! ! character!from!t=0.9! () () The!memory!function,! M t ,!is!equal!to! Sν t !at!high!temperatures!and!in!the! ! ! linear!response!regime.!Most!probe/solvent!systems!have!a!linear!response!because! excitation!of!the!chromophore!usually!results!in!a!small!change!in!the!dipole! moment!of!the!molecule!and!therefore!effects!a!small!perturbation!to!the!motions! and!structure!of!the!solvent!molecules.18,19! ! Figure!1.3.!Dynamic!Stokes!shift!of!the!ZnCytc!fluorescence!spectrum.!Reprinted! with!permission!from!Lampa‑Pastirk!and!Beck,!2006,!American!Chemical!Society.! The!response!shows!an!initial!red‑shift!followed!by!a!blue‑shift!of!fluorescence!for! ZnCytc!in!water!at!room!temperature!following!excitation!in!the!Soret!band!(blue)! and!a!red‑shift!following!excitation!of!the!Q‑band!(red).!In!each!case,!the!center! frequency!of!the!0‑0!transition!was!plotted!as!a!function!of!time!delay!after! excitation.20! ! 10! By!using!three!solvents!to!vary!viscosity!and!polarity,!water,!a!water/glycerol! mixture,!and!a!water/methanol!mixture,!the!Beck!Lab!was!able!to!compare!effects! on!the!FSS.!In!water,!there!is!an!initial!red!shift,!then!a!blue!shift.!At!increased! viscosity,!the!initial!red!shift!is!smaller,!the!turn‑around!is!delayed,!and!the!blue! shift!is!slower.!At!lower!viscosity,!there!is!half!the!blue!shift!relative!to!pure!water,! no!initial!red!shift,!and!a!final!red!shift.!It!is!likely!that!the!initial!red!shift!is!not! visible!at!low!viscosity!because!it!occurs!on!a!time!scale!shorter!than!can!be! observed!with!the!instrument!that!was!used.!The!final!red!shift!that!is!observed!at! low!viscosity!is!not!observed!at!high!viscosity,!presumably!because!it!occurs!too!late! to!be!detected.!Overall,!there!are!three!phases,!an!initial!red!shift!that!includes! unfolding!motions!and!solvent!entering!the!hydrophobic!core,!a!blue!shift!when!the! structure!is!comparable!to!the!thermally!unfolded!equilibrium!state,!and!a!final!red! shift!that!occurs!during!refolding!and!relaxation!back!to!the!native!state.20! There!are!two!main!and!independent!factors!that!affect!the!three!phases.! Activation!energy!barrier!height!and!hydrodynamic!friction!both!affect!the! transition!between!phases!and!are!completely!independent!of!each!other.!Lower! polarity!corresponds!to!lower!activation!energy!barrier!height.!Lower!viscosity! corresponds!to!lower!solvent!friction.!Friction!between!the!solvent!and!exposed! peptides!contributes!directly!to!the!kinetic!control!of!the!product!state.!The! observations!made!by!the!Beck!Lab!suggest!that!the!transition!state!more!closely! resembles!the!unfolded!state!than!the!native!state!and!that!perturbation!by! temperature!or!solution!composition!involves!macroscopic!state!changes.20! ! 11! It!is!thought!that!the!vibrational!energy!is!transferred!from!the!chromophore!to! the!protein!by!intramolecular!vibrational!redistribution!(IVR)!through!Cys14!and! Cys17,!not!through!the!binding!site!peptides.11!Though!the!vibrational!energy!is! ultimately!transferred!to!the!solvent,!transfer!to!the!solvent!is!slow,!on!the!order!of! 20!ps,!which!allows!the!protein!to!be!affected!by!the!vibrations.!These!effects!can! result!in!small!structural!changes,!leading!to!stages!of!unfolding.20!The!data!showed! an!unusual!bidirectional!response!with!excitation!of!the!Soret!band!and!a! biexponential!shift!in!the!time‑resolved!fluorescence!spectrum!when!excited!at! 584nm!(see!Figure!1.3).!The!fast!component!is!thought!to!be!random!motions!of!the! hydrophobic!core.!The!slow!component!is!due!to!motions!of!the!protein!in!areas! contacting!solvent.20! 1.2.3!Intramolecular!Vibrational!Excitation! Intramolecular!Vibrational!Excitation!(IVE)!experiments!in!the!Beck!Lab!have! used!optically‑driven!excitation!from!the!ground!state!into!multiple!vibrational! levels!above!the!first!excited!state.20!If!there!is!energy!in!excess!of!the!gap!between! the!S0!and!S1!states!that!optically!excites!a!chromophore,!the!excited!molecule!will! relax!to!the!lowest!vibrational!state!of!the!first!excited!electronic!state!before! relaxing!to!the!ground!state!by!emitting!a!photon!or!sometimes!relaxes!vibrationally! before!transferring!excitation!to!another!chromophore!in!the!photosynthetic! system.!The!vibrational!relaxation!occurs!by!transferring!energy!to!the!surrounding! media,!in!this!case,!a!protein.!The!transfer!of!excess!vibrational!energy!to!the!protein! occurs!within!2‑4!ps.21,22!The!protein!ultimately!transfers!the!vibrational!energy!to! ! 12! the!surrounding!solvent.20!IVE!experiments!present!an!integrated!response!rather! than!the!time‑resolved!response!shown!in!the!aforementioned!studies.! E Sn A S1 F S0 ! Figure!1.4.!!Electronic!and!vibrational!energy!levels!as!found!in!molecules!like!the! porphyrin!of!ZnCytc.!Reprinted!with!permission!from!Lampa‑Pastirk!and!Beck,! 2006,!American!Chemical!Society.!After!an!initial!optical!excitation!(blue),! vibrational!relaxation!to!the!S1!state!occurs!(green),!followed!by!fluorescence! emission!(red).20! ! ! 13! A S1 F S0 ! Figure!1.5.!Absorption!(A)!and!fluorescence!(F)!dipole!strength!spectra!obtained!at! room!temperature.!Reprinted!with!permission!from!Lampa‑Pastirk!and!Beck,!2006,! American!Chemical!Society.!The!vibrational!structure!of!the!Q‑band!absorption!and! fluorescence!spectra!is!due!to!the!0‑0!and!0‑1!vibronic!transitions.20! As!described!earlier,!an!ensemble!of!folding!trajectories!would!be!expected! based!on!the!protein‑folding!funnel!potential!energy!surface!hypothesis.!The!native! state!of!the!protein!is!expected!to!be!at!the!lowest!point!on!the!potential!energy! surface.23,24!Research!using!IVE!performed!by!the!Beck!Lab!has!shown!that!an! ensemble!of!folding!trajectories!may!not!accurately!describe!the!ZnCytc!system! because!the!results!were!interpreted!in!terms!of!a!specific!pathway!between!three! partially!unfolded!states.20! The!time‑resolved!fluorescence!spectrum!of!ZnCytc!exhibits!a!conventional!shift! to!the!red!with!excitation!in!the!Q!absorption!band.!This!shift!occurs!as!vibrational! ! 14! energy!is!transferred!from!the!porphyrin!to!the!protein.!As!the!protein!acquires! increased!vibrational!energy,!it!begins!to!unfold.!The!time!scale!of!IVR!is!thought!to! be!long!enough!for!partial!unfolding!to!occur.!By!changing!the!excitation! wavenumber,!the!amount!of!vibronic!energy!transferred!to!the!protein!can!be! varied.!IVE!of!the!protein!structure!results!in!at!least!three!partially!unfolded!states! along!a!specific!pathway.!Along!this!pathway,!three!main!transitions!are!observed!in! both!ZnCytc!and!fbCytc.!There!is!an!initial!red!shift,!then!a!blue!shift,!and!a!shift!back! to!the!red,!though!the!net!shift!is!blue.25! The!transitions!of!the!IVE!profile!(Figure!1.6A),!which!appear!to!mark!activation! enthalpies!for!displacements!of!the!native!structure!to!partially!unfolded!states,! occur!at!roughly!the!same!position!as!the!transitions!of!the!!F0−1 / F0−0 !plot!(Figure! 1.6B)!and!also!those!of!the!Soret!band.!Figure!1.6C!shows!that!there!is!overall! narrowing!of!the!0‑0!peak,!but!the!change!is!small,!less!than!5%,!while!local!melting! would!be!expected!to!broaden!the!line!width!as!excitation!shifted!blue.25! ! ! 15! Dipole Strength 550 Wavelength (nm) 450 400 500 350 Soret Q 3 -1 0–0 (10 cm ) 0 17.045 A 17.040 F0–1/F0–0 17.035 1.7 B 1.6 1.5 1.4 -1 0–0 (cm ) C 204 202 200 198 196 0 2 4 8 6 3 cm-1) ex- 0–0 (10 10 12 14 ! Figure!1.6.!Dependence!of!the!fluorescence!spectrum!from!ZnCytc!at!22°C!on!the! intramolecular!vibrational!excitation:!(A)!the!integrated!Stokes!shift,!as!determined! by!the!wavenumber!of!the!0‑0!peak!maximum,!!ν 0−0 ;!(B)!ratio!of!the!dipole! strengths!of!the!0‑1!and!0‑0!peaks,!!F0−1 / F0−0 ;!and!(C)!the!half‑width!at! half‑maximum!of!the!0‑0!peak,!!Δν 0−0 .!The!plotted!abscissa!is!the!intramolecular! vibrational!excitation,!the!difference!between!the!excitation!wavenumber!and!that! of!the!0‑0!vibronic!transition.!At!the!top!of!the!figure,!the!absorption!dipole!strength! spectrum!is!plotted!with!respect!to!the!same!abscissa!scale.!The!vertical!dashed! lines!mark!the!excitation!energies!corresponding!to!apparent!activation!enthalpies! for!three!protein‑unfolding!transitions.!Reprinted!with!permission!from!Barns$et$al.,! 2008,!American!Chemical!Society.25! ! 16! 0.7 F01 Fi / Σ F i 0.6 0.5 F00 0.4 0.3 0 2 4 6 8 10 12 0 2 4 6 8 10 12 1.8 1.6 F01 / F00 1.4 1.2 1.0 0.8 0.6 0.4 Delay (ns) ! Figure!1.7.!Time!evolution!of!the!dipole!strengths!for!the!0‑1!and!0‑0!vibronic! transitions!obtained!from!time‑resolved!fluorescence!spectra!from!ZnCytc!in!water! (22!°C).!Reprinted!with!permission!from!Lampa‑Pastirk!et$al,!2004,!American! Chemical!Society.10!Top:!Peak!intensity!normalized!by!total!dipole!strength.!Bottom:! Ratio!of!peak!intensities.!! ! 17! As!the!solvent!environment!around!the!porphyrin!is!exposed!to!more!aqueous! solvent,!it!will!exhibit!an!increase!in!polarity!resulting!in!the!initial!red!shift.!In! fbCytc,!there!are!no!metal‑protein!axial!interactions!as!found!in!ZnCytc,!so!a! relatively!lower!barrier!height!is!expected.!The!absence!of!metal!at!the!center!of!the! porphyrin!in!fbCytc!leads!to!an!unstable!molecule.25!Similarly,!the!six‑coordinate! interaction!with!zinc!is!strained!relative!to!native!ferric!cytochrome!c!(FeCytc),!and! therefore!less!stable!than!the!native!protein.!The!strained!interaction!in!ZnCytc! results!in!an!early!dissociation!of!Met80,!on!a!time!scale!of!about!100!ps.10!The!ratio! of!!F0−1 !to!!F0−0 !is!a!measure!of!axial!ligand!coordination.!Earlier!work!showed!that! the!intensity!of!the!!F0−0 !and!!F0−1 !peaks!change!with!respect!to!time!(see!Figure! 1.7).10!The!rapid!change!in!!F0−1 / F0−0 !occurs!when!there!is!a!dramatic!increase!in! His18!dissociation.!The!dissociation!rate!is!limited!by!nonpolar!reorganization!of! solvent!molecules.!The!second!structural!transition!is!a!blue!shift,!which! accompanies!a!decrease!in!polarity!surrounding!the!porphyrin.!Blue!shifts!are! generally!associated!with!transitions!from!native!states!to!molten‑globule!or! acid‑denatured!states.!This!transition!is!comparable!to!thermal!unfolding.!As!this! reorganization!occurs,!different!ligands!may!bind!axially!to!the!porphyrin.!The!third! structural!transition!is!another!red!shift,!which!again!indicates!an!increased! exposure!of!the!porphyrin!to!the!polar!solvent.!The!third!transition!is!likely!to!be!a! ligand‑exchange!in!ZnCytc.25! The!detection!of!a!sequence!of!activation!barriers!demonstrates!steps!along!an! unfolding!pathway.25!The!results!were!inconsistent!with!a!Maxwell‑Boltzmann! distribution,!which!would!predict!a!distribution!over!all!states!on!the! ! 18! protein‑folding!potential!energy!surface.5!Instead,!partially!unfolded!states!appear! in!a!sequential!manner!and!eventually!refold.25! 1.2.4!Relevance!to!Dynamics!in!LH!and!RC!Proteins!in!Photosynthesis! While!the!previous!discussion!of!protein!dynamics,!folding,!and!vibrational! energy!dissipation!is!relevant!to!proteins!in!general,!it!is!also!particularly!relevant!to! proteins!involved!in!photosynthesis!where!energy!absorbed!by!chlorophyll! molecules!is!stored!in!terms!of!charge‑transfer!reactions!across!membranes.!In!the! work!described!here,!ZnCytc!is!treated!as!a!model!for!a!one‑chlorophyll‑binding! protein!and!shares!a!mode!of!binding!to!the!chromophores!in!the!photosynthetic! reaction!center!(RC).!! It!allows!insight!into!light!harvesting!and!how!excess!light!energy!may!be! dissipated!from!the!chromophore!to!the!surroundings.! Photosynthetic!organisms!use!light‑harvesting!proteins26!that!contain! chlorophyll!and!carotenoid!chromophores!to!convert!solar!energy!to!chemical! energy.27!Each!kind!of!chromophore!is!able!to!collect!energy!in!a!specific!range!of! wavelengths!by!absorbing!a!photon!that!initiates!a!π!to!π*!transition.!The!excitation! energy!is!eventually!transferred!to!a!photosynthetic!reaction!center!(RC)28,29! where!it!drives!electron‑transfer!reactions!and!generates!a!transmembrane!redox! potential!gradient.27!The!potential!gradient!drives!phosphorylation!of!adenosine! diphosphate!to!yield!adenosine!triphosphate.30! Over!the!last!four!decades,!X‑ray!crystal!structures!have!been!determined!for! many!photosynthetic!light‑harvesting!and!reaction!center!proteins!giving!an! ! 19! accurate!picture!of!the!location!and!relative!arrangement!of!the!chromophores.31R39! An!important!example!of!a!light!harvesting!protein!is!LHCII,!the!major! light‑harvesting!complex!associated!with!photosystem!II!in!higher!plants.!LHCII! consists!of!a!trimeric!assembly!of!protein!subunits!with!C3‑symmetry.!Each!subunit! contains!its!own!chromophores,!including!chlorophylls!(8!Chl!a!and!6!Chl!b)!and! carotenoids!(Lut!1,!Lut!2,!Neo,!and!Vio!with!Lut:Neo:Vio=2:1:0.5).40,41!The! arrangement!of!the!chromophores!optimizes!energy!transfer!from!chlorophyll$b! (Chl!b)!to!chlorophyll!a!(Chl!a).42!Chl!a!molecules!act!as!the!terminal!emitters!that! mediate!energy!transfer!to!the!reaction!center.43!Energy!transfer!between!the! chromophores!found!in!light!harvesting!proteins!can!proceed!by!one!of!two! mechanisms.!Excitation!delocalization!is!a!process!that!allows!coherent!energy! transfer!and!involves!strong!coupling!between!molecules!that!are!have!small!spatial! separation!and!aligned!dipole!moments.42!Förster!resonance!energy!transfer! (FRET)!occurs!when!there!is!weak!coupling!between!the!transition!dipole!moments! of!two!chromophores.44!Carotenoids!function!as!accessory!light!harvesting! chromophores!that!allow!absorption!of!wavelengths!that!cannot!be!absorbed!by! chlorophyll!molecules41!and!also!provide!protection!against!photodamage!by! trapping!triplet!excited!states.45!LHCII!arranges!the!chromophores!to!collect!solar! energy!of!many!wavelengths!and!optimize!energy!transfer!to!the!RC.29,46!The! arrangement!of!chromophores!within!LHCII!avoids!non‑radiative!quenching!of! excitation,!though!it!is!unclear!how!this!is!accomplished.!Under!high‑light!conditions! when!excess!excitation!cannot!be!transferred!to!the!reaction!center!and!could!cause! cellular!damage,!the!system!initiates!non‑photochemical!quenching!(NPQ)!which! ! 20! harmlessly!dissipates!excess!excitation!energy.!NPQ!could!be!mediated!by!small! changes!in!protein!conformation!that!change!the!distances!and!orientations!of!the! chromophores!in!relation!to!one!another.47! In!addition!to!optimizing!light!harvesting!and!energy!transfer,!LHCII!may!serve! as!a!dynamic!solvent!medium!for!the!chromophores!rather!than!a!rigid!scaffold.! Protein!dynamics!may!play!an!important!role!in!reorganizing!chromophores.!The! Beck!group!is!interested!in!understanding!how!motion!of!a!protein!structure! responds!to!the!formation!and!decay!of!excited!states!resulting!in!an!optimization!of! energy!transfer!or!energy!storage.9,48!The!main!focus!of!recent!studies!has!been!on! dynamic!solvation,!which!is!defined!as!the!reorganization!of!solvent!molecules!in! response!to!a!change!in!electronic!state!or!charge!state!of!a!solute!chromophore.49! In!the!case!of!chromoproteins,!it!describes!the!impact!of!conformational!changes! and!structural!fluctuations!of!a!surrounding!protein!on!intrinsic!chromophores!and! their!energy!levels.9,20,25,48!For!intrinsic!chromophores,!the!protein!and!associated! liquid!solvent!molecules!act!together!as!the!solvent.48!When!a!solute!such!as!a! chlorophyll!molecule!absorbs!visible!light,!a!π!to!π*!transition!occurs!which!changes! the!dipole!moment!of!the!chromophore.!A!change!in!the!dipole!moment!is!likely!to! affect!the!protein!structure!as!the!protein!and!surrounding!solvent!shift!to!the! minimum!of!the!new!potential!energy!surface!and!relax!into!a!new!equilibrium! state.10,50!These!changes!are!expected!to!occur!for!all!excited!solute/solvent!pairs! including!those!where!a!protein!acts!as!the!solvent.!LHCII!is!only!one!example!of!a! protein!that!may!rearrange!in!response!to!chromophore!excitation.51!To!avoid! complications!in!data!analysis!for!many!chromophore!systems,!it!is!desirable!to! ! 21! study!protein!dynamics!in!a!simple!system!that!will!give!a!more!basic!understanding! of!the!chromophore/protein!interactions!upon!excitation.! The!Beck!Lab!has!studied!the!model‑system!ZnII‑substituted!cytochrome!c! (ZnCytc)!in!the!past.!ZnCytc!is!a!good!model!system!because!it!contains!a!single! fluorescent!intrinsic!chromophore.!This!avoids!the!complications!of!a! many‑chromophore!system.!The!main!questions!addressed!in!the!ZnCytc!work! pertain!to!the!timescales!and!character!of!the!protein!and!solvent!motion!that!occur! in!response!to!the!formation!of!the!ZnII!porphyrin!excited!state.9,20!When!an! intrinsic!chromophore!within!a!protein!is!excited,!it!introduces!perturbations!to!the! surrounding!protein.!Those!perturbations!are!thought!to!disturb!the!native!fold!of! the!protein!as!energy!is!dissipated!and!ultimately!transferred!to!surrounding!water! molecules.9,20,48!Since!these!motions!may!include!structural!transitions!from!the! native!structure!to!partially!unfolded!structures,!the!results!may!also!contribute!to! understanding!how!cytochrome!c!unfolds!and!refolds.9,20,25!Current!understanding! of!protein!folding!can!be!described!by!a!series!of!conformational!changes!along!a! funnel‑shaped!potential!energy!surface.!The!funnel‑shaped!potential!energy!surface! leads!to!the!lowest‑energy!state,!known!as!the!native!state.3!The!native!state!of!a! protein!is!the!arrangement!of!the!primary!structure!into!secondary!and!tertiary! structures,!primarily!driven!by!hydrophobic!and!hydrophilic!interactions!of!the! amino!acid!side!chains.52,53!Along!the!potential!energy!surface,!there!are!a!series!of! activation‑energy!barriers.1,24,52!As!a!protein!folds,!it!moves!from!local!minimum!to! local!minimum!by!climbing!over!activation‑energy!barriers!that!are!thermally! accessible.53,54!As!a!protein!is!unfolded!from!the!native!state,!it!would!be!expected! ! 22! to!follow!a!reverse!set!of!trajectories.!Instead,!the!results!suggest!that!the!protein! transitions!into!a!discrete!set!of!conformations!rather!than!randomly!sampling!the! protein‑folding!funnel.9,25!Understanding!the!response!of!a!protein!to!excitation!of! a!chromophore!could!result!in!a!more!accurate!picture!of!the!ways!photosynthetic! proteins!are!able!to!efficiently!transfer!energy!in!some!circumstances!and!divert! energy!away!from!the!reaction!center!in!others.!The!way!a!protein!dissipates! vibrational!energy!as!the!excitation!is!transferred!between!chromophores!toward! the!reaction!center!may!play!a!central!role!in!its!function.! ! ! ! 23! CHAPTER!2! ! Light‑driven!Partial!Unfolding!of!ZnII‑substituted! Cytochrome!c! Summary! The!nature!of!the!partially!unfolded!structures!that!are!generated!in! ZnII‑substituted!cytochrome!c!(ZnCytc)!upon!optical!excitation!above!the!vibronic! origin!of!the!Q!(S0→S1,!π→π*!transition)!band!was!investigated!using! continuous‑wave!fluorescence!spectroscopy.!The!excess!vibrational!energy! prepared!in!the!S1!state!is!transferred!by!intramolecular!vibrational!redistribution! on!the!<2!ps!timescale!to!the!protein!surroundings.!This!excitation!of!the!protein! results!in!structural!transitions!that!change!the!environment!of!the!ZnII!porphyrin! and!shift!the!fluorescence!spectrum.!Step‑like!transitions!of!the!fluorescence! spectrum's!Stokes!shift!correspond!to!the!activation!threshold!for!changes!in! structure!from!the!native!state!to!a!partially!unfolded!state!associated!with!the!Ω! loop!formed!by!residues!20–35,!which!is!adjacent!to!the!Cys14!and!Cys17!thioether! linkages!from!the!porphyrin!to!the!polypeptide!backbone.!This!structural! assignment!is!based!on!studies!of!the!excitation‑wavelength!dependence!of!the! fluorescence!Stokes!shift!as!a!function!of!temperature!and/or!the!presence!of! guanidinium!ions!(Gdm⁺),!either!of!which!allows!a!distinction!between!the! responses!of!the!native!and!globally!unfolded!states.!The!excitation!energy!for! optical!formation!of!the!unfolded!state!is!consistent!with!the!previous! ! 24! determination!by!Englander!and!coworkers!using!hydrogen‑exchange!NMR! spectroscopy!in!ferricytochrome!c!in!the!presence!of!Gdm⁺.! 2.1!Introduction! The!energy!landscape/protein‑folding!funnel!hypothesis!suggests!that!a!partially! unfolded!structure!can!descend!to!the!native!state!along!a!range!of!distinct! trajectories!rather!than!a!discrete!pathway!of!intermediate!states.1R4,24,52,55,56! Beck!and!coworkers!suggested!that!this!picture!could!be!tested!by!following!the! propagation!of!partially!unfolded!structures!after!optical!excitation!from!the!native! state.48!In!ZnII‑substituted!cytochrome!c!(ZnCytc),!it!was!demonstrated!that!at!least! three!partially!unfolded!structures!were!obtained!after!excitation!of!the!intrinsic! ZnII!porphyrin!(see!Figure!2.1)!well!above!the!S1!(Q!band)!0–0!transition! (Figure!2.2);!the!excess!vibrational!energy!provides!the!activation!energy!for! unfolding!reactions!from!the!native!state.25!This!approach!was!termed! intramolecular$vibrational$excitation!(IVE).!By!scanning!the!wavelength!of!the!light! source,!the!vibrational!excitation!provided!to!the!protein!by!radiationless!decay!and! intramolecular!vibrational!redistribution!(IVR)!could!be!continuously!tuned.!After! the!vibrational!energy!transfer!process!is!completed,!the!fluorescence!emission!of! the!vibrationally!cooled!S1‑state!ZnII!porphyrin!could!then!be!used!as!a!probe!of!the! protein‑refolding!process.!! ! 25! ! ! Figure!2.1.!Ribbon!(left)!and!solvent‑excluded!surface!(right)!renderings!of!the! X‑ray!crystal!structure!of!horse‑heart!ferricytochrome!c (1hrc.pdb).11!The! porphyrin!and!associated!structures!(axial!ligands:!His18!and!Met80!and!thioether! linkages:!Cys14!and!Cys17)!are!shown!as!stick!structures!in!the!ribbon!picture.!The! protein!structure!is!color!coded!from!red!to!blue!in!order!of!relative!folding!stability! following!the!scheme!of!Englander!and!coworkers:57!residues!70–85!(red),!residues! 36–61!(yellow),!residues!20–35!and!the!α!helix!over!residues!60–70!(green),!and! the!N‑!and!C‑terminal!α!helices!(blue).!Based!on!a!comparison!of!2D!NMR!spectra,! ZnCytc!is!isostructural!with!the!native,!FeII‑containing!protein!in!solution.58,59! The!nature!of!the!partially!unfolded!structures!produced!in!ZnCytc!by!the!IVE! process!was!left!indeterminate.25!The!fluorescence!spectrum!from!the! ZnII!porphyrin!exhibits!step!transitions!of!the!integrated!Stokes!shift!and!vibronic! structure!when!the!excitation!is!tuned!above!the!threshold!that!generates!an! unfolded!state.!The!integrated!Stokes!shift!provides!information!on!changes!in!the! protein!and!solvent!structure!that!surrounds!the!ZnII!porphyrin,!whereas!the! vibronic!structure!provides!information!on!the!presence!or!absence!of!protein!or! solvent‑derived!axial!ligands!to!the!ZnII!ion.!Because!the!fluorescence!lifetime!in! ZnCytc!is!~4!ns,!the!changes!in!structure!that!are!detected!in!terms!of!the!Stokes! ! 26! shift!and!vibronic!structure!of!the!fluorescence!spectrum!are!likely!to!be!short! ranged!in!character,!but!it!is!not!clear!from!the!previous!work!whether!local!or! global!structural!rearrangements!are!involved.!Because!the!excitation!wavelengths! that!yield!unfolded!structures!are!near!to!the!onset!of!the!Soret!(S0!→!Sn)!absorption! band,!however,!there!is!additionally!the!question!of!whether!photochemical! processes!involving!the!axial!ligands!of!the!ZnII!porphyrin48!are!precursors!to!the! unfolding!reaction.!! ! 27! E Sn S4 S3 Soret S2 Q ISC S1 T1 A F P S0 ! Figure!2.2.!Jablonski!energy‑level!diagram!describing!photophysical!processes!in! ZnCytc.!Vibronic!energy!levels!are!represented!by!horizontal!lines.!Absorption!of!a! photon!is!represented!by!the!vertical!blue!arrow.!Nonradiative!relaxation!(NR)!and! intersystem!crossing!(ISC)!are!indicated!by!wavy!arrows.!Ground‑state!recovery!via! fluorescence!(F)!and!phosphorescence!(P)!is!indicated!by!the!green!and!red!arrows,! respectively;!additional!nonradiative!recovery!processes!to!the!ground!state!are!not! shown.!! In!this!chapter,!these!questions!are!addressed!by!new!experiments!in!which!the! IVE!response!sensed!by!the!fluorescence!spectrum!of!ZnCytc!is!characterized!as!a! function!of!temperature!and!in!the!presence!of!guanidinium!ions!(Gdm+).!If!the! structural!changes!in!ZnCytc!are!driven!by!vibrational!excitations,!an!increase!in!the! ! 28! temperature!of!the!medium!surrounding!the!protein!would!be!expected!to!result!in! a!lowering!of!the!activation!threshold!for!an!unfolding!reaction.!The!optical!energy! required!to!drive!the!native!state!to!the!unfolded!state!would!be!decreased!as!the! temperature!increases!because!the!vibrational!energy!arising!from!the! Maxwell‑Boltzmann!distribution!would!be!added!to!the!vibrational!energy!obtained! by!IVR!from!the!absorbed!photon.!If!the!IVE!response!involves!just!the!local! environment!of!the!ZnII!porphyrin,!it!would!be!completely!transformed!by! denaturing!the!protein!at!high!temperatures!or!in!the!presence!of!Gdm+.! The!results!strongly!suggest!that!the!unfolding!reactions!driven!by!IVE!in!ZnCytc! involve!changes!in!conformation!of!the!local!polypeptide!region!adjacent!to!the! thioether!linkages!to!the!ZnII!porphyrin.!This!conclusion!is!supported!by!the! observation!that!the!denatured!protein!exhibits!an!even!larger!IVE‑induced! response!than!the!native!protein!and!by!a!determination!of!the!heat!capacity!of!the! affected!protein!region.!The!likely!origin!of!the!IVE!response!is!shown!to!be!a! conformational!change!in!the!omega!loop!identified!by!the!hydrogen‑exchange!NMR! experiment!by!Englander!and!coworkers57!as!the!highest!energy!protein!folding! unit!(foldon)!along!the!cytochrome!c!unfolding!pathway.! 2.2!Experimental!Section! 2.2.1!Sample!Preparation.!ZnCytc!was!prepared!from!horse‑heart! ferricytochrome!c!(Sigma)!using!the!procedure!described!by!Vanderkooi.50!Liquid! anhydrous!hydrogen!fluoride!(Linde)!was!used!as!the!demetalating!agent.!The! reaction!was!run!on!a!home‑built!gas‑handling!system!in!Teflon!reaction!vessels.! ! 29! The!metal‑free!or!free‑base!protein!(fbCytc)!was!isolated!using!strong!cation! ion‑exchange!chromatography!on!a!Whatman!CM‑52!column.!Reconstitution!with! ZnII!was!performed!at!50!°C!in!the!presence!of!a!10‑fold!molar!excess!of!zinc!acetate! (Sigma!379786‑5G,!99.999%).!Completion!of!the!demetallation!and! metal‑reconstitution!reactions!were!determined!spectrophotometrically!by! observing!differences!in!the!number!and!position!of!bands!in!the!Q!band!region!of! the!absorption!spectrum.! The!ZnCytc!product!solution!was!further!prepared!using!methods!described!by! Winkler!and!coworkers60!and!by!Kostić!and!coworkers.61!The!protein!solution!was! desalted!and!the!protein!was!isolated!by!cation‑exchange!chromatography!on!a! clean!Whatman!CM‑52!column.!Fractions!containing!ZnCytc!were!equilibrated!with! 25!mM!sodium!phosphate!buffer!at!pH!7.0!by!repeated!concentration!over!an! Amicon!YM‑10!ultrafiltration!membrane!(Millipore)!and!subsequent!dilution!with! the!buffer!solution.!The!final!product!was!concentrated,!aliquoted,!flash‑frozen,!and! stored!at!–80!°C!prior!to!use.! For!use!in!fluorescence!experiments,!ZnCytc!samples!were!thawed!and!diluted!in! 25mM!pH!7.0!sodium!phosphate!buffer!solution!to!obtain!an!absorbance!of!0.15– 0.17!for!the!Q!band!maximum.!For!Gdm+!experiments,!the!dilution!solution!was! prepared!to!contain!the!desired!concentration!of!Gdm+!and!25!mM!sodium! phosphate!buffer!at!pH!7.0!when!a!sample!was!diluted!to!3.0!mL.!The!absorption! spectrum!was!recorded!after!dilution!of!each!sample!in!a!1!cm!quartz!cuvette!using! an!Ocean!Optics!USB2000!spectrometer!(2!nm!bandpass)!and!a!Mikrotechnic! DH‑2000!fiber‑optic!light!source.! ! 30! 2.2.2!Continuous‑wave!Absorption!and!Fluorescence!Spectroscopy.! Absorption!spectra!were!acquired!with!a!Hitachi!U‑4001!spectrophotometer!(2!nm! bandpass).!Fluorescence!spectra!were!obtained!using!a!home‑built!fluorescence! spectrometer!consisting!of!a!Jobin‑Yvon!AH10!100!W!tungsten‑halogen!light!source,! a!Jobin‑Yvon!H10!excitation!monochromator!(4!nm!bandpass),!an!Acton!Research! SP‑150!emission!spectrograph!(2!nm!bandpass),!and!a!Jobin‑Yvon!Symphony!CCD! detector.!The!CCD!detector!employs!a!liquid!nitrogen!cooled,!back‑illuminated,! 2000!⨉!800!pixel!silicon!detector!chip!(EEV!corporation).!A!300!groove/mm! diffraction!grating!(500!nm!blaze!wavelength)!was!mounted!in!the!emission! spectrograph,!resulting!in!a!270!nm!spectral!range!imaged!over!2000!vertically! binned!channels!on!the!CCD!detector!chip.!As!presented!as!a!function!of! wavenumber,!the!fluorescence!intensities!are!multiplied!by!the!square!of!the! wavelength!in!order!to!compensate!for!the!fixed!(in!wavelength!units)!spectral! bandpass!of!the!emission!spectrograph.!The!absorption!and!fluorescence! instruments!are!controlled!by!LabVIEW!(National!Instruments)!programs.!! For!IVE!experiments,!the!temperature!of!the!sample!cuvette!was!maintained!by!a! Quantum!Northwest!TLC50F!Peltier!effect!temperature!controller!at!temperatures! ranging!from!5!°C!to!90!°C.!The!wavelength!of!the!excitation!light!source!was! calibrated!over!the!entire!scan!range!for!an!IVE!experiment!and!daily!at!the!584!nm! wavelength!of!the!0–0!peak!of!the!ZnCytc!absorption!spectrum!by!measuring!the! spectrum!at!the!sample!position!with!an!Ocean!Optics!USB2000!spectrometer.!The! Ocean!Optics!spectrometer!was!calibrated!using!a!mercury!emission!spectrum.!The! spatially!integrated!power!of!the!excitation!beam,!as!estimated!using!a!Coherent! ! 31! Fieldmaster!power!meter!and!associated!silicon!photodiode!detector,!was!10! µ W!at! 522.9!nm.!Fluorescence!emission!spectra!were!acquired!as!the!average!of!typically! twenty!60‑second!exposures!of!the!CCD!detector.! 2.3!Results! 2.3.1!Dependence!of!Fluorescence!Spectra!on!Vibrational!Excitations.!As! reported!in!previous!work,!the!fluorescence!spectrum!from!ZnCytc!shifts!as!the! excitation!light!source!is!tuned!above!the!wavenumber!of!the!0–0!vibronic! transition!owing!to!structural!transitions!of!the!surrounding!protein.!At!low! excitation!wavenumbers,!the!fluorescence!spectrum!shown!in!Figure!2.3!is! observed;!the!intensity!of!the!spectrum!scales!with!the!relative!absorption!as!the! excitation!wavenumber!is!scanned,!but!the!shape!and!the!position!of!the!spectrum! are!unchanged.!The!excitation!wavenumber!chosen!for!this!spectrum,!19120!cm−1! (522.9!nm),!is!2,000!cm−1!above!that!for!the!0–0!vibronic!transition,!where!the! absorption!and!fluorescence!spectra!cross.!The!shift!to!the!red!of!the!0–0!peak!of!the! fluorescence!spectrum!from!that!of!the!absorption!spectrum!provides!an!estimate!of! 57!cm−1!for!the!solvation!reorganization!energy,! λ ,! ! ! ( ) λ = ν 0#0,A − ν 0#0,F /2 ! ! (2.1)! This!shift!corresponds!to!the!time!integral!of!the!dynamic!fluorescence!Stokes!shift,! the!time‑dependent!stabilization!of!the!excited!state!that!occurs!following!optical! excitation!of!the!ZnII!porphyrin!arising!from!polar!and!nonpolar!reorganizational! motions!of!the!surrounding!protein!and!solvent.9,10!In!the!absence!of!a!net! ! 32! vibrational!excitation,!the!protein!and!solvent!response!is!triggered!only!by!the! ground‑to‑excited!state!changes!in!permanent!dipole!moment!and!size!of!the!ZnII! porphyrin.!! At!significantly!higher!net!vibrational!excitations,!>4,000!cm−1,!the!fluorescence! Stokes!shift!and!the!vibronic!structure!of!the!fluorescence!spectrum!exhibit!changes! that!arise!from!discrete!transitions!of!the!protein!structure!(see!Figures!2.4!and!2.5).! The!absorption!dipole!strength!is!continuously!non‑zero!at!excitation!wavelengths! below!600!nm!(see!Figure!2.3),!so!the!net!vibrational!excitation!provided!to!the! protein!by!IVR!from!the!ZnII!porphyrin!can!be!varied!at!least!to!10,000!cm−1!by! scanning!the!excitation!light!source.!(At!excitation!wavelengths!above!450!nm,!a! range!of!excited!singlet!states!Sn,!!n ≥ 2 ,!are!excited;!nonradiative!decay!to!the!S1! state!occurs!on!the!<50!fs!timescale!in!ZnII!porphyrins.62)!Analysis!of!the!set!of! spectra!shown!in!Figure!2.4!illustrate!the!changes!that!evidence!transitions!in! protein!structure.!The!fluorescence!Stokes!shift!exhibits!an!oscillatory!response;!the! entire!fluorescence!spectrum!shifts!first!to!the!red,!then!to!the!blue,!and!finally!back! to!the!red!as!the!excitation!wavenumber!is!scanned!over!the!4,000–10,000!cm−1! range.!The!magnitudes!of!these!red!and!blue!shifts!are!less!than!the!solvation! reorganization!energy,!so!they!are!small!relative!to!the!wavenumber!range!shown!in! the!set!of!spectra!in!Figure!2.4,!but!a!first‑derivative!peak‑maximum! determination25!for!the!0–0!peak!over!a!larger!set!of!spectra!obtains!the!profile! shown!in!Figure!2.5a.!Representative!derivative!functions!were!shown!in!previous! work!by!the!Beck!group.25!Synchronous!with!these!shifts!is!an!oscillation!in!the! relative!intensities!of!the!0–0!and!0–1!fluorescence!peaks!(see!Figure!2.4!and!2.5b).!! ! 33! Wavelength (nm) 700 650 600 550 500 450 400 Soret Dipole Strength 0-1 0-1 Q 0-0 A/ν F/ν3 x12 15000 20000 25000 -1 Frequency (cm ) ! Figure!2.3.!Continuous!wave!absorption!(blue)!and!fluorescence!(red)! dipole‑strength!spectra!from!ZnCytc!at!20!°C!and!pH!7.0,! A ν /ν !and! F ν /ν 3 ,! ! ! respectively.!The!absorbance!and!fluorescence!spectra!were!normalized!to!the! amplitude!of!the!0–0!peak.!The!fluorescence!spectrum!was!excited!at!523!nm! (19120!cm−1).!The!S0→S1!vibronic!transition!is!located!where!the!two!spectra!cross,! at!587.2!nm!(17030!cm−1).! () ! 34! () 1.5 A 523 nm B 452 nm 0-0 1.0 0.5 0 Relative Dipole Strength 1.5 0-0 1.0 0.5 0 1.5 1.0 C 425 nm 0-0 D 408 nm 0-0 0.5 0 1.5 1.0 0.5 0 14000 16000 18000 -1 Frequency (cm ) ! Figure!2.4.!Fluorescence!dipole‑strength!spectra!from!ZnCytc!at!30!°C!with! excitation!at!(a)!523!nm!(!ν IVE = 2000 cm −1 ),!(b)!452!nm!(!ν IVE = 5000 cm −1 ),! (c)!425!nm!(!ν IVE = 6350 cm −1 ),!and!(d)!408!nm!(!ν IVE = 7350 cm −1 ).!The!same! integration!time!was!used!in!each;!the!spectra!are!plotted!with!normalization!to!the! amplitude!of!the!0–0!peak.!The!spectrum!shown!in!(a)!is!the!same!spectrum!shown! in!Figure!2.3.!! ! 35! In!the!following!narrative,!we!will!call!the!dependence!of!the!wavenumber!of!the! peak!maximum!of!the!0–0!peak!in!the!ZnCytc!fluorescence!spectrum!(as!in! Figure!2.5a)!an!IVE$profile.!The!corresponding!plot!of!the!ratio!of!the!intensities!of! the!0–1!and!0–0!peaks,!!F0$1 /F0$0 ,!will!be!called!an!FR$profile!(Figure!2.5b).!Both! profiles!are!expressed!with!respect!to!!ν IVE ,!the!net!vibrational!excitation!above!the! 0–0!transition,!! ! !ν IVE = ν ex − ν 0(0 ! ! (2.2)! As!shown!in!Figure!2.5,!there!are!three!step!transitions!observed!in!the!FR!and!IVE! profiles!over!the!4,000–10,000!cm−1!range.!These!transitions!(I,!II,!and!III)!can!be! described!by!sigmoidal!functions!of!the!form! ! ! ( A ⎛ ⎛ erf x − x 0 F x = y0 + ⎜ ⎜ 2⎜⎜ 2σ ⎝⎝ ! () ) ⎞ +1⎞ ⎟ ⎟ ⎟ ⎠ ⎟ ⎠ ! (2.3)! which!are!integrals!of!Gaussian!distributions!with!width! σ !and!center!wavenumber! !x 0 ;!the!other!parameters!are!introduced!in!the!inset!to!Figure!2.5a.!The!first! transition!(I)!accompanies!a!shift!of!the!fluorescence!spectrum!to!the!red;! transitions!II!and!III!are!associated!with!blue!shifts.!A!smoothing!spline!was!used!to! describe!nonparametrically!the!final!region!of!the!IVE!profile!(IV),!which! accompanies!a!shift!of!the!fluorescence!spectrum!to!the!red,!because!the!character! of!this!part!of!the!IVE!and!FR!profiles!varies!with!experimental!conditions!and!is!not! well!described!by!a!single!transition.! ! 36! Excitation Wavelength (nm) 500 450 425 2σ II I A 16950 F0-1/F0-0 375 III 16960 1.8 400 IV A -1 ν0-0 (cm ) 16970 475 y0 x0 B 1.6 II I 2000 IV III 1.4 4000 6000 8000 10000 -1 νIVE (cm ) ! Figure!2.5.!Evolution!of!the!continuous‑wave!fluorescence!spectrum!from!ZnCytc!at! 30!°C!as!a!function!of!!ν IVE ,!the!excitation!energy!above!the!wavenumber!of!the! S1 state!vibronic!origin:!(a)!wavenumber!of!the!peak!dipole!strength!in!the!0–0!peak! and!(b)!intensity!ratio!for!the!0–1!and!0–0!peaks,!!F0#1 / F0#0 .!The!data!is!divided!into! four!sections!including!transitions!I,!II,!and!III,!which!were!superimposed!with! models!described!by!Equation!2.3,!and!section!IV,!which!has!a!smoothed!curve! drawn!through!the!data.!(Inset)!Parameters!for!the!sigmoidal!functions! (Equation!2.3)!used!to!describe!step!transitions!in!the!peak!wavenumber!and! fluorescence!ratio!profiles:!!x 0 ,!the!center!wavenumber!of!the!transition;!! y 0 ,!the! value!of!the!function!prior!to!the!transition;!!A ,!the!amplitude!of!the!transition;!and! σ ,!the!width!of!the!transition.! The!structural!nature!of!the!shifts!of!the!fluorescence!spectrum!of!ZnCytc!that! accompany!the!transitions!in!the!IVE!profile!is!not!clear!just!from!a!consideration!of! ! 37! the!magnitudes!of!the!shifts.!The!shifts!to!the!red!(transition!I,!20!cm−1)!and!blue! (transitions!II!and!III,!28!cm−1!total)!are!half!as!large!as!the!solvent!reorganization! energy,!so!one!might!conclude!that!the!transitions!in!the!IVE!response!are! associated!with!small!reorganizations!of!protein!and!solvent!around!the!ZnII! porphyrin.!It!should!be!noted,!however,!that!the!fluorescence!spectrum!shifts! comparably!to!the!blue!when!ZnCytc!is!denatured!thermally!or!in!the!presence!of! Gdm+;!a!shift!of!20!cm–1!is!observed!as!the!temperature!increases!over!the!70–90!°C! range,!and!a!shift!of!52!cm−1!is!observed!in!the!presence!of!4!M!Gdm+.!Because!the! ground‑to‑excited‑state!change!in!permanent!dipole!moment!and!molecular!size!are! small,!the!ZnII!porphyrin!is!a!relatively!insensitive!probe!of!protein!and!structural! dynamics.!As!shown!below,!however,!we!can!use!the!temperature!and!Gdm+! dependence!of!the!IVE!and!FR!profiles!to!structurally!define!the!IVE!transitions.! The!FR!profiles!provide!information!on!protein‑limited!rates!of!change!of!the! ZnII!porphyrin's!conformation!following!photodissociation!of!its!axial!ligands.25!The! relative!dipole!strength!of!the!0–0!and!0–1!peaks!in!the!fluorescence!spectra!of!ZnII! porphyrins!reports!changes!in!the!conformation!of!the!porphyrin!that!arise!from! binding!of!axial!ligands.63R66!Based!on!resonance!Raman!spectral!evidence,!Kostić! and!coworkers61!conclude!that!the!ZnII!porphyrin!in!ZnCytc!assumes!a! five‑coordinate!configuration!like!that!preferred!by!ZnII!porphyrins!in! solution64,65,67,68,!as!opposed!to!the!six‑coordinate!structure!found!in!crystals!of! native!ferricytochrome!c!(see!Figure!2.1).11!If!correct,!this!assignment!suggests!that! the!methionine,!Met80,!does!not!make!a!significant!interaction!with!the!ZnII!ion.! Owing!to!packing!forces!in!molecular!crystals,!however,!a!number!of!strained! ! 38! six‑coordinate!ZnII!porphyrins!have!been!observed!in!X‑ray!diffraction!studies.69R71! Owing!to!the!evidence!from!ZnII/FeII‑substituted!cytochrome!c!co‑crystals72!and! from!2D!NMR!spectroscopy58,59!that!ZnCytc!assumes!a!structure!that!is! isomorphous!with!that!of!FeCytc,!it!is!known!that!the!Met80!and!His18!side!chains! attack!the!ZnII!ion!in!ZnCytc!in!ligand‑binding!configurations.!We!suggest!at!this! point!that!the!ZnII!porphyrin!in!ZnCytc!adopts!a!strained!six‑coordinate!structure! with!a!weaker!interaction!with!the!Met80!side!chain!(2.30!Å!from!metal!center)! than!with!the!His18!side!chain!(2.16!Å!from!metal!center)11.!Both!of!these!axial! ligands!photodissociate!in!the!S1!state,!yielding!a!time!profile!for!the!!F0$1 /F0$0 !ratio! that!increases!at!rates!that!are!associated!with!reorganization!of!the!protein!around! the!expanded!cavity!required!by!the!ZnII!porphyrin.10!Thus,!as!the!!F0$1 /F0$0 !ratio! determined!from!the!continuous‑wave!fluorescence!spectrum!rises!(transition!I),! the!rate!of!the!protein!response!to!release!of!the!axial!ligands!increases!compared!to! the!fluorescence!timescale.!A!decrease!in!the!!F0$1 /F0$0 !ratio!(transitions!II!and!III)! indicates!that!ligands!are!rebinding!to!the!ZnII!ion.!This!portion!of!the!FR!profile!was! previously!assigned!to!rebinding!of!a!different!histidine!side!chain!or!a!water! molecule,!accounting!for!a!decrease!in!the!!F0$1 /F0$0 !ratio!during!transitions!II!and! III!in!an!FR!profile.20,25!It!should!be!noted!that!the!shape!of!the!FR!profile!observed! in!the!previous!work!is!significantly!different!than!that!shown!in!Figure!2.5b;!the! !F0$1 /F0$0 !ratio!exhibits!a!much!smaller!increase!in!transition!IV!in!the!present! work.! In!the!following,!we!report!how!the!IVE!and!FR!profiles!depend!on!the!sample! temperature!and!Gdm+!concentration!in!an!effort!to!determine!whether!the!step! ! 39! transitions!we!observe!correspond!to!local!or!global!structural!transitions!and!to! determine!their!mechanism!of!formation.!A!key!observation!is!that!the!denatured! state!exhibits!IVE!and!FR!profiles!that!are!similar!in!shape!to!those!observed!in!the! native!state!but!that!the!magnitude!of!the!IVE!shifts!are!much!larger!in!the! denatured!state.! 2.3.2!Temperature!Dependence!of!the!IVE!and!FR!Profiles.!Figure!2.6! compares!the!IVE!profiles!of!ZnCytc!solutions!over!the!5–90!°C!temperature!range.! Each!profile!is!represented!in!the!figure!by!sigmoidal!transitions!(Equation!2.3)!or!a! smoothing!spline!(for!transition!IV)!fit!to!the!measured!values!of!the!peak! wavenumber!of!the!0–0!peak!as!a!function!of!!ν IVE .!The!supporting!materials!include! separate!plots!like!Figure!2.5!for!each!temperature!showing!the!measured!data! points!and!the!fitted!curves.!As!the!temperature!increases,!the!fluorescence! spectrum!observed!with!low!!ν IVE !shifts!initially!to!the!red!over!a!26!cm−1!range,!but! above!70!°C!the!spectrum!shifts!to!the!blue!over!a!18!cm−1!range.!This!reversal! marks!the!thermal!denaturation!of!the!protein.73!The!shape!of!the!IVE!profiles! observed!above!and!below!70!°C!are!comparable,!but!the!total!range!of!the!shift! from!red!to!blue!over!transitions!I–III!increases!markedly!in!the!denatured!state.! ! 40! 16980 5°C 16940 70°C -1 ν0-0 (cm ) 16960 A 17000 16980 90°C 16960 16940 70°C B 2000 4000 6000 8000 10000 -1 νIVE (cm ) ! Figure!2.6.!IVE!profiles!for!ZnCytc!as!a!function!of!temperature.!Curves!at!each! temperature!were!obtained!by!fitting!the!experimental!peak!wavenumbers! (Equation!2.3).!The!integrated!Stokes!shift,!as!determined!by!the!wavenumber!of!the! 0–0!peak!maximum,!!ν 0#0 .!Peak!maxima!are!plotted!as!points.!The!trendline!includes! a!fit!using!Equation!2.3!to!describe!each!of!the!first!three!apparent!transitions!in!the! data!and!a!smoothed!curve!to!describe!the!data!after!the!peak.!(a)!The!integrated! Stokes!shift!at!eight!temperatures!(5!°C,!10!°C,!20!°C,!30!°C,!40!°C,!50!°C,!60!°C,!and! 70!°C)!demonstrating!the!change!in!the!profile!as!temperature!increases!to!the! denaturation!point!of!the!protein.!(b)!The!integrated!Stokes!shift!at!four! temperatures!(70!°C,!80!°C,!85!°C,!and!90!°C),!demonstrating!the!change!in!the! profile!as!temperature!increases!above!the!denaturation!point!of!the!protein.!! Figure!2.7!shows!the!corresponding!FR!profiles!over!the!same!range!of!sample! temperatures.!The!midpoint!of!transition!I!does!not!vary!significantly!with! temperature;!the!precision!of!the!midpoint!determinations!here!and!in!the!IVE! profiles!is!±50!cm−1.!The!same!is!true!for!transitions!II!and!III!below!70!°C,!but!at! ! 41! higher!temperatures!a!modest!blue!shift!is!observed.!The!average!value!of!the! !F0$1 /F0$0 !ratio!decreases!as!the!temperature!increases,!but!note!that!the!amplitude! of!transitions!I–III!gets!larger!as!the!temperature!increases.!These!results!suggest! that!the!reorganizational!response!to!the!photodissociation!of!the!ZnII!porphyrin's! axial!ligands!generally!slows!as!the!temperature!increases!but!that!the!rate! increases!markedly!over!the!!ν IVE !range!associated!with!transitions!I–III.!The!latter! observation!reflects!our!conclusion!that!transitions!I–III!involve!excited‑state! exchange!of!axial!ligands;25!transition!I!is!associated!with!a!release!of!ligands! whereas!transitions!II!and!III!are!associated!with!rebinding!of!ligands.! F0-1 / F0-0 2.0 1.6 5°C 1.2 90°C 2000 4000 6000 νIVE (cm-1) 8000 10000 ! Figure!2.7.!Dependence!of!the!fluorescence!peak!ratio!on!intramolecular!vibrational! excitation!at!multiple!temperatures.!A!line!graph!for!each!temperature!shows!the! actual!data!points!obtained!after!smoothing!the!fluorescence!curve!and!using!a! peak‑finding!routine!to!identify!the!peak!position!and!intensity.!! ! 42! Figure!2.8!compares!the!temperature!dependences!of!the!midpoints,! x 0,i ,!of!the! ! transitions!in!the!IVE!and!FR!profiles.!For!transitions!I!and!II!(Figure!2.8a!and!8b,! respectively),!the!data!points!in!the!native!and!denatured!states!are!described!by! separate!linear!trendlines.!The!intersection!of!the!trendlines!provides!an!estimate! for!the!denaturation!temperature,!65!°C.!Transitions!I!and!II!exhibit!a!small!initial! red!shift!followed!by!a!sharp!blue!shift!once!the!protein!denatures.!Transition!III! (Figure!2.8c),!in!contrast,!exhibits!an!initial!blue!shift!and!reaches!a!constant!value! above!30!°C.!The!midpoints!of!the!FR!profile!transitions!are!essentially!independent! of!temperature!below!the!denaturation!temperature.! The!negative!slopes!of!the!plots!shown!in!Figure!2.8!for!the!IVE!profile!midpoints! for!transitions!I!and!II!suggest!an!interpretation!as!heat!capacities!for!the!portion!of! the!system!that!is!undergoing!a!transformation!during!the!IVE!transitions:! ! ! dx dε − 0≅ = Cv ! ! dT dT (2.4)! The!rate!of!change!of!the!midpoint!of!a!transition,!!x 0,i ,!with!respect!to!temperature,! !T ,!is!equated!to!the!rate!of!change!of!the!average!energy!of!the!system,! ε ,!which!is! formally!equal!to!the!heat!capacity!at!constant!volume.74!This!equation!is!suggested! by!the!hypothesis25!that!the!vibrational!energy!obtained!via!IVR!of!the!excess! excitation!energy!above!the!S1!state!provides!the!activation!enthalpy!required!for!a! change!in!structure!of!the!protein.!As!the!sample!temperature!increases,!the!mean! internal!energy!of!the!system!increases!according!to!the!Maxwell‑Boltzmann! distribution,!so!the!extra!energy!from!the!photon!that!is!required!to!reach!the! ! 43! activation!threshold!for!a!protein!structural!transition!should!exhibit!a!weak! negative!temperature!dependence.!The!behavior!shown!in!Figures!2.8a!and!2.8b!for! transitions!I!and!II!is!generally!consistent!with!this!expectation.!The!magnitude!of! the!slopes!(3.4!cm−1!K−1=!9.7!cal!mol−1!K−1!and!1.5!cm−1!K−1=!4.3!cal!mol−1!K−1)! are!very!small!compared!to!the!overall!heat!capacity!(4192!cal!mol−1!K−1!.75)!This! finding!raises!the!suggestion!that!the!activation!thresholds!are!not!constant!with! respect!to!temperature,!but!it!seems!clear!that!the!structural!transitions!are!local!to! the!protein!region!connected!to!the!ZnII!porphyrin!rather!than!involving!the!whole! protein.! Above!the!thermal!denaturation!temperature,!the!IVE!profile!midpoints!for! transitions!I!and!II!exhibit!positive!slopes.!These!results!suggest!that!the!residual! structure!in!the!denatured!state!evolves!with!temperature.!The!comparable!positive! slope!associated!with!transition!III!at!low!temperatures!in!Figure!2.8c!reports!that! the!affected!region!of!the!protein!obtains!a!structure!similar!to!that!in!the!denatured! state.! ! 44! C -1 x0,I (cm ) -1 x0,II (cm ) -1 x0,III (cm ) 7000 6500 B 6000 5500 A 5000 4500 0 10 20 30 40 50 60 70 80 90 Temperature (°C) ! Figure!2.8.!Temperature!dependences!of!the!midpoints,!!x 0,i ,!for!the!IVE!and!FR! profiles!from!ZnCytc:!(a)!Transition!I;!(b)!Transition!II;!(c)!Transition!III.!For!(a)!and! (b),!the!trendlines!shown!at!low!temperatures!includes!data!points!ranging!from!5°C! to!50°C;!the!trendlines!shown!at!higher!temperatures!includes!data!points!from! 70°C!to!90°C.!For!(c),!the!trendline!shown!at!low!temperatures!includes!data!points! ranging!from!5°C!to!30°C;!the!trendline!at!higher!temperatures!includes!data!points! from!30°C!to!90°C.! Figure!2.9!compares!the!temperature!dependences!of!the!amplitudes,!!Ai ,!of!the! transitions!in!the!IVE!and!FR!profiles.!These!plots!report!the!change!in!the! environment!of!the!ZnII!porphyrin!that!accompanies!the!red!and!blue!shifts!of!the! fluorescence!spectrum.!Below!the!denaturation!temperature,!the!red!shift! associated!with!transition!I!(Figure!2.9a)!and!the!sum!of!the!blue!shifts!associated! with!transitions!II!and!III!(Figure!2.9b)!are!essentially!constant.!Above!the! ! 45! denaturation!temperature,!the!red!and!blue!shifts!increase!significantly!as!the! temperature!increases.!These!results!make!it!clear!that!the!structural!change! associated!with!the!IVE!transitions!is!a!change!in!the!conformation!of!the!part!of!the! protein!that!packs!around!the!ZnII!porphyrin!in!the!native!state!and!in!the! denatured!state.!Further,!the!affected!portion!of!the!protein!makes!a!larger! structural!change!when!excited!by!IVE!in!the!denatured!state!than!in!the!native! state.!! -1 |A2+3| (cm ) 80 B 60 40 -1 |A1| (cm ) 20 40 A 20 0 10 20 30 40 50 60 Temperature (°C) 70 80 90 ! Figure!2.9.!Absolute!value!of!the!amplitudes!of!the!first!red!shift,!!A 1 ,!and!the!blue! shift,!!A 2+3 ,!of!the!IVE!profile!as!a!function!of!temperature.!For!(a)!and!(b),!the! trendlines!shown!at!low!temperatures!includes!data!points!ranging!from!5°C!to! 70°C!(slope:!0.003!cm–1/°C!and!0.054!cm–1/°C,!respectively),!below!the!protein’s! transition!to!a!denatured!state.!The!trendlines!shown!at!higher!temperatures! includes!data!points!from!70°C!to!90°C!(slope:!1.03!cm–1/°C!and!2.18!cm–1/°C,! respectively),!after!the!protein!is!denatured.!! ! 46! 2.3.3!Gdm+!Dependence!of!the!IVE!Profile.!Figure!2.10!shows!the!variation!of! the!IVE!profile!of!ZnCytc!as!Gdm+!is!added!to!the!solution.!Sigmoidal!transitions! (Equation!2.3)!or!a!smoothing!spline!(for!transition!IV)!represent!the!trends!in!each! profile!(see!the!supporting!materials!for!the!data!points!and!fitted!curves).!At!low! !ν IVE ,!the!fluorescence!spectrum!shifts!to!the!blue!according!to!a!two‑state! denaturation!transition!with!a!midpoint!at!1.75!M!Gdm+!(!ΔGFold = −10.74 !kJ!mol−1,! !m = 6.16 !kJ!mol−1;!Jagnya!Tripathy,!manuscript!in!preparation).!The!IVE!transitions! substantially!increase!in!amplitude!as!the!denaturation!transition!occurs! (1-3!M!Gdm+)!just!as!observed!for!the!thermal!denaturation!of!the!protein.!Even!at! low!Gdm+!concentrations,!however,!the!midpoints!of!the!transitions!progressively! shift!to!higher!!ν IVE !as!Gdm+!is!added.! -1 ν0-0 (cm ) 17070 17040 4M 17010 16980 16950 0M 4000 6000 8000 10000 -1 νIVE (cm ) ! Figure!2.10.!Dependence!of!the!IVE!profile!of!ZnCytc!as!a!function!of!!ν IVE !on!Gdm+! concentration.!The!integrated!Stokes!shift!at!seven!concentrations!of!guanidinium! ion!(0.0!M,!1.0!M,!1.5!M,!2.0!M,!2.5!M,!3.0!M,!and!4.0!M),!demonstrating!the!change!in! the!profile!as!denaturant!concentration!increases!until!the!protein!is!fully! denatured.! ! 47! Figures!2.11!and!2.12!plot!the!Gdm+!dependences!of!the!midpoints,! x 0,i ,!and! ! amplitudes,!!Ai ,!of!the!IVE!profile!transitions.!Unlike!the!temperature!dependence! (Figure!2.9),!the!midpoints!of!the!IVE!transitions!(Figure!2.11)!do!not!exhibit!a!clean! break!between!the!native!and!denatured!states!as!Gdm+!is!added!(estimated! uncertainty!in!data!points!is!approximately!100!cm−1).!Instead,!the!profiles!exhibit! roughly!linear!responses,!indicating!that!the!activation!thresholds!for!the!IVE! transitions!substantially!increase!as!Gdm+!binds!to!exterior!sites!on!the!part!of!the! protein.!In!contrast,!the!amplitudes!(Figure!2.12)!show!distinct!regions!for!the! native!and!denatured!states!as!the!Gdm+!concentration!is!varied.!The!largest! amplitudes!for!transition!I!(Figure!2.12a)!and!for!the!sum!of!the!blue!shifts!in! transition!II!and!III!(Figure!2.12b)!are!observed!at!the!end!of!the!unfolding! transition!(2.5!M).!This!behavior!is!comparable!to!that!observed!in!the!thermal! denaturation!of!the!protein;!the!change!in!the!ZnII!porphyrin's!environment!that! accompanies!the!IVE!transitions!is!much!larger!in!the!denatured!state.!The!region!of! the!protein!that!is!affected!by!the!IVE!transitions!is!apparently!more!constrained!in! the!native!state!than!in!the!denatured!state.!The!decrease!in!the!amplitudes!shown! in!Figure!2.12!above!the!denaturation!concentration!may!involve!binding!of!Gdm+! to!regions!of!the!protein!that!were!protected!in!the!native!state.! ! 48! 6700 6500 C B -1 -1 x0,II (cm ) x0,III (cm ) 7000 6000 -1 x0,I (cm ) 5500 5500 A 5000 4500 0 1 2 3 4 5 + [Gdm ] (M) ! Figure!2.11.!The!excess!excitation!wavenumber!midpoint,!!x 0 ,!of!each!transition!as! a!function!of!guanidinium!concentration,!(a)!!x 0,I ,!(slope:!201!cm–1/M)!(b)!!x 0,II ,! (slope:!137!cm–1/M)!(c)!!x 0,III ,!(slope:!34!cm–1/M).! ! 49! 120 B |A2+3| (cm-1) 100 80 60 40 |A1| (cm-1) 20 40 A 20 0 1 2 3 + [Gdm ] (M) 4 5 ! Figure!2.12.!Absolute!value!of!the!amplitude!of!the!red!shift!of!the!ZnCytc! fluorescence!spectrum!in!transition!I,!!A 1 ,!and!the!sum!of!the!amplitudes!for!the! blue!shift,!!A2 + A3 ,!in!the!IVE!profile!as!a!function!of!guanidinium!ion!concentration.! For!both!(a)!and!(b),!the!trendlines!shown!at!low!Gdm+!concentration!include!data! points!ranging!from!0.0!M!to!1.0!M!(slope:!2.3!cm–1/°C!and!18.3!cm–1/°C,! respectively),!below!the!protein’s!transition!to!a!denatured!state.!The!trendlines! shown!at!higher!Gdm+!concentration!includes!data!points!from!2.5!M!to!5.0!M! (slope:!–4.5!cm–1/°C!and!–9.1!cm–1/°C,!respectively),!after!the!protein!is!denatured.! 2.4!Discussion! The!experiments!described!in!this!paper!provide!an!improved!picture!for!the! nature!of!the!structural!changes!that!accompany!excitation!of!the!intrinsic!ZnII! porphyrin!in!ZnCytc!above!its!0–0!transition!to!the!S1!state.!The!main!conclusion!is! that!a!small!section!of!the!protein!undergoes!a!conformational!change!owing!to! ! 50! being!excited!by!the!excess!vibrational!energy!it!obtains!via!IVR!from!the!ZnII! porphyrin.!This!conclusion!is!required!because!the!IVE!profile!exhibits!a! characteristic!red–blue–red!shape!in!the!native!state!and!in!the!denatured!states! produced!thermally!or!by!addition!of!Gdm+.!The!vibrational!nature!of!the!excitation! is!confirmed!by!the!lowering!of!the!activation!threshold!for!transitions!I!and!II!as!the! temperature!is!raised.!Further,!the!portion!of!the!IVE!profile!associated!with!a!blue! shift!of!the!fluorescence!spectrum!(transitions!II!and!III)!is!the!part!of!the!profile! that!yields!an!unfolded!character.! Because!the!IVE!transitions!are!driven!by!IVR‑mediated!transfer!of!energy!from! the!ZnII!porphyrin!to!the!polypeptide!backbone!in!ZnCytc,!the!regions!of!the!protein! that!are!involved!in!IVE‑driven!conformational!changes!are!nearby!to!the!Cys14!and! Cys17!thioether!linkages!(see!Figure!2.13).!The!wavenumber!regions!associated! with!transitions!I–III!span!the!wavelength!region!between!the!Q!and!Soret!bands,! which!are!formally!porphyrin‑centered!!π → π * !transitions.!The!most!efficient! channels!for!IVR!from!the!porphyrin!to!the!protein!are!through!covalent!bonds,! through!the!thioether!linkages!of!Cys14!and!Cys17.!These!two!cysteines!are!on!one! end!of!the!amino‑terminal!blue!region!(residues!1!through!19),!adjacent!to!the!green! loop!(residues!20!through!35)!in!the!Englander!color!scheme!(see!Figures!2.1!and! 2.13).57! ! 51! Cys17 Cys14 ! Figure!2.13.!Another!view!of!the!X‑ray!crystal!structure!of!horse‑heart! ferricytochrome!c (1hrc.pdb)11!following!the!scheme!of!Englander!and! coworkers.57!The!figure!has!been!rotated!when!compared!to!Figure!2.1!to!allow!a! view!of!the!two!thioether!linkages,!Cys14!and!Cys17.!Also!note!the!blue!dot! representing!an!intrinsic!water!molecule!adjacent!to!the!porphyrin.!! A!reasonable!assignment!for!the!regions!of!the!ZnCytc!protein!that!undergo! partial!unfolding!during!transitions!II!and!III!can!be!obtained!from!a!comparison!of! the!activation!thresholds!determined!from!the!IVE!transitions!and!the!Gibbs! reaction!energies!for!activation!of!the!cooperative!protein‑folding!groups!identified! by!Englander!and!coworkers.57!Equilibrium!hydrogen‑exchange!NMR!(HX‑NMR)! experiments!with!FeCytc!in!the!presence!of!Gdm+!determined!the!Gibbs!energies!of! reaction!for!the!opening!of!different!parts!of!the!protein!so!that!exchange!of!protons! with!the!surrounding!aqueous!solvent!is!accelerated.!These!cooperative!units57!or! foldons76R78!are!shown!in!Figure!2.1;!the!two!most!stable!regions!of!the!protein!are! ! 52! associated!with!the!regions!colored!blue!and!green!(at!30!°C,!!ΔG = 53.6 !kJ/mol!and! 41.8!kJ/mol,!respectively).!The!IVE!experiment!effectively!results!in!a!vertical! excitation!of!the!affected!regions!of!the!protein;!the!excitation!occurs!on!a!short! timescale!compared!to!protein!and!solvent!motion,!so!the!associated!change!in! structure!is!instantaneously!small!(!ΔS ≈ 0 ).25!Thus,!the!!ΔH !associated!with!the!IVE! event!at!constant!temperature!can!be!approximately!equated!to!a!Gibbs!energy!of! activation,!!ΔG ‡ :! ! ! ( ) ΔG ‡ = ΔH ‡ −T ΔS ‡ ≈ 0 ≅ ΔH ‡ ! ! (2.5)! Using!this!relationship,!the!midpoint!of!transition!I!corresponds!to! ‡ !ΔG = 51.8 !kJ/mol!at!30!°C!(see!Figure!2.8,!!x 0,I = 4330 !cm−1).!This!value!is!not! greater!than!that!reported!by!Englander!and!co‑workers!for!the!process!that! promotes!exchange!of!protons!in!the!blue!region.!The!reaction!profile!for!an! endoergic!(!∆G > 0 )!change!in!structure!would!be!expected!to!consist!of!a! non‑negligible!excess!Gibbs!energy!between!the!transition!and!product!states!that! corresponds!to!!∆G ‡ !for!the!reverse!reaction.!The!difference!in!Gibbs!energy! between!!∆G ‡ !and!!∆G !is!significant!for!the!green!loop,!10.0!kJ/mol,!so!it!remains!a! candidate!for!assignment!to!transition!I.!Because!transition!I!corresponds!to!a!red! shift!of!the!fluorescence!spectrum,!however,!it!is!assigned!to!a!conformational! change!of!the!native!state!that!is!not!unfolding!in!character,!so!it!may!be!that!the! HX–NMR!experiments!would!not!detect!it.!In!contrast,!!ΔG ‡ !at!30!°C!for!transitions!II! and!III!are!66.4!kJ/mol!(!x 0,II = 5550 !cm−1)!and!81.5!kJ/mol!(!x 0,III = 6812 !cm−1),! ! 53! respectively.!These!activation!energies!are!compatible!with!structural!assignments! for!unfolding!processes!to!the!blue!region!or!the!green!region!that!would!be! expected!to!result!in!deprotected!regions!of!the!protein!and!promote!hydrogen! exchange.!! The!thermodynamic!analysis!given!above!is!supported!by!the!structural! criterion!that!the!affected!protein!regions!must!be!sequence‑adjacent!to!the!Cys14! and!Cys17!thioether!linkages!in!order!to!be!efficiently!excited!by!IVR.!A!second! requirement!is!that!the!affected!regions!must!be!adjacent!to!the!ZnII!porphyrin!in! the!native!structure!so!that!shifts!of!the!fluorescence!spectrum!might!be!expected! when!an!IVE!transition!occurs.!These!requirements!favor!assignment!of!transitions! II!and!III!to!the!green!loop.!The!green!loop!begins!three!amino!acids!after!Cys17!and! provides!most!of!the!native!state!environment!to!the!His18‑face!of!the!ZnII! porphyrin,!where!the! π !electrons!would!sense!changes!in!environment!that!shift! the!fluorescence!spectrum.!The!blue!region!leading!from!Cys14!to!the!N‑terminus!is! distant!from!the!ZnII!porphyrin!in!the!native!fold.!The!short!loop!between!Cys14!and! Cys17!is!unlikely!to!be!flexible!owing!to!its!restriction!by!the!thioether!linkages!to! the!porphyrin!macrocycle,!so!conformational!changes!of!that!region!are!unlikely!to! contribute!to!the!IVE!transitions.!! 2.5!Conclusions! We!have!characterized!the!step!transitions!exhibited!in!the!integrated! fluorescence!Stokes!shift!of!ZnCytc!that!occur!in!IVE!profiles,!the!dependence!of!the! fluorescence!Stokes!shift!on!the!excitation!wavenumber!above!the!0–0!transition!of! ! 54! the!S1!state.!These!transitions!evidence!conformational!(transition!I)!and!partial! unfolding!(transitions!II!and!III)!reactions.!A!comparison!of!the!activation! thresholds!for!transitions!II!and!III!with!the!Gibbs!energy!of!unfolding!for!the! cooperative!units!detected!by!Englander!and!coworkers!with!HX–NMR!spectroscopy! suggests!a!structural!assignment!to!the!green!omega!loop!adjacent!to!Cys17!in!the! amino‑acid!sequence.!The!through‑bond!flow!of!vibrational!energy!from!the!ZnII! porphyrin!to!the!polypeptide!backbone!by!IVR!processes!promotes!the!unfolding! reactions!in!the!IVE!experiment.$ These!findings!are!significant!because!they!support!the!idea!that!a!range!of! partially!unfolded!structures!are!microscopically!populated!during!spontaneous! protein!folding.!The!vertical!excitation!resulting!from!IVE!provides!the!means!to! drive!a!protein!from!the!native!state!to!a!range!of!partially!unfolded!states!provided! that!an!electronic!chromophore!can!provide!sufficient!activation!energy.!The! present!results!suggest!propagation!of!vibrational!energy!through!the!polypeptide! backbone!is!limited!in!travel!by!the!rate!at!which!vibrational!energy!is!dissipated!to! contacting!regions!of!the!protein!or!to!the!surrounding!solvent.!It!is!possible!that!the! dissipation!of!vibrational!energy!that!accompanies!downhill!excitation!energy! transfer!in!photosynthetic!light‑harvesting!proteins!is!associated!with!the! IVE‑driven!conformational!transitions!that!affect!the!energy‑transfer!yield!in! nonphotochemical!quenching!and!other!photoregulatory!responses.! ! ! ! ! 55! CHAPTER!3! Solvation!Dynamics!of!the!Hydration!Shell!of!ZnII‑Substituted! Cytochrome!c1! ! Summary! The!hydration!shell!of!ZnII‑substituted!cytochrome!c!(ZnCytc)!was!probed!using! the!indolecyanine!dye!Cy5!using!femtosecond!pump–continuum‑probe! spectroscopy.!Cy5!was!attached!to!a!surface!lysine!residue!by!a!flexible!linker;!the! distance!between!the!surface!of!the!protein!and!the!chromophore!was! approximately!10!Å.!Because!Cy5's!lowest!energy!π→π*!transition!does!not!result!in! a!change!in!permanent!dipole!moment,!the!solvation!response!measured!by!the!rate! at!which!the!stimulated!emission!or!excited‑state!absorption!features!in!the!pump– probe!spectrum!shift!to!the!red!or!blue,!respectively,!can!be!treated!approximately! using!the!viscoelastic!continuum!theory!described!previously!by!Berg!and! coworkers.79!Further,!the!damping!of!excited‑state!wavepacket!motions!along! out‑of‑plane!coordinates!provides!an!additional!measurement!of!the!solvent! viscosity.!The!main!conclusion!of!this!work!is!that!the!hydration!shell!is!as!much!as! 160!times!more!viscous!than!bulk!water.!A!simple!structural!interpretation!of!this! finding!is!that!the!hydration!shell!of!ZnCytc!is!polarized!by!surface!charges!and!by! interruption!of!the!long‑range!hydrogen!bonding!network!by!the!folded!protein! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 1!This!work!was!done!in!collaboration!with!Michael!Bishop!and!Jerome!D.!Roscioli.! ! 56! solute!so!that!longer!or!more!persistent!chains!of!hydrogen‑bonded!water! molecules!are!present!than!in!the!bulk.! 3.1!Introduction! The!hydration!shell!of!a!protein!constitutes!a!domain!of!"biological!water"80R83! that!contributes!to!the!stability!of!the!native!folded!structure84R87!and!to!its! biological!function,!especially!in!charge!transfer!or!redox!catalysis,88!as!a!result!of! its!distinct!dynamical!properties!compared!to!the!bulk.89R92!Gruebele!and! coworkers!used!tetrahertz!spectroscopy!to!probe!the!dynamics!of!the!hydration! shell!by!varying!the!concentration!of!a!protein!solution;!they!determined!that!the! hydration!shell!has!distinct!properties!that!extend!at!least!1.0!nm!from!the!protein! surface.93!Zewail!and!coworkers81,82,94!used!femtosecond!time‑resolved! fluorescence!measurements!with!probe!chromophores!on!or!tethered!to!a!protein's! surface!to!show!that!the!diffusive!(or!random!reorganizational)!part!of!the!polar! solvation!response!observed!using!the!dynamic!fluorescence!Stokes!shift!(FSS)!is! slowed!at!least!to!the!10–100!ps!timescale!from!the!sub‑ps!regime!that!is! characteristic!of!bulk!water.95!Reorientational!dynamics!slow!comparably!in!water! near!interfaces,96,97!but!the!solvation!response!detected!by!intrinsic!or! protein‑bound!probes!reaches!well!into!the!nanosecond!regime.6,48,98R103!! Considering!that!changes!in!the!observed!timescale!follow!the!protein‑folding! state,!the!nanosecond!part!of!the!solvation!response!in!proteins!can!be!primarily! attributed!to!protein‑derived!motions.9!The!contribution!of!the!hydration!shell!to! the!detected!solvation!response,!however,!is!not!easily!distinguished!from!that!of! ! 57! the!protein.19,88,104,105!In!fact,!molecular!dynamics!simulations!by!Matyushov!and! coworkers!suggest!that!the!motions!of!water!molecules!in!the!hydration!shell!on!the! nanosecond!timescale!arise!from!electrostatic!interactions!with!surface!charges!and! mechanical!coupling!(or!"slaving"54,106)!to!conformational!motions!of!the!solvated! protein.88,107R110!! The!long!polar!solvation!timescales!observed!in!the!hydration!shell!were! modeled!phenomenologically!by!Bagchi!and!coworkers!using!a!dynamic!exchange!of! water!molecules!between!the!hydration!shell!and!binding!sites!on!the!protein! surface.82,83!The!Bagchi!model!holds!that!the!structure!of!the!hydration!shell!is! equivalent!to!that!of!bulk!water;!the!slow!timescales!arise!from!long!residence!times! on!the!protein!surface.!Halle91!has!pointed!out!that!the!solvation!dynamics!results! and!Bagchi's!exchange!model!are!in!conflict!with!the!results!of!17O!NMR!relaxation! dispersion!(MRD)!experiments!and!of!molecular!dynamics!simulations!of!the! dielectric!response!of!a!protein!and!its!surrounding!solvent,!which!suggest!that!the! motions!of!water!molecules!in!the!hydration!shell!are!only!somewhat!slowed! compared!to!those!in!the!bulk.19!Halle!and!Davidovic90,91!suggested!that!the! apparent!disagreement!between!the!FSS!and!MRD!experiments!might!be!resolved!if! the!viscosity!of!the!hydration!shell!is!larger!than!that!of!bulk!water.!Because!the! viscosity!is!a!long‑range!or!collective!property!of!medium,!Halle's!picture!requires! that!the!water!molecules!in!the!hydration!shell!be!organized!differently!than!in!the! bulk.! One!approach!to!test!these!hypotheses!involves!measuring!the!viscosity!of!the! hydration!shell!of!a!protein!using!a!nonpolar!electronic!probe.!The!change!in!a! ! 58! probe's!electronic!structure!that!accompanies!optical!excitation!usually!results!in!a! change!in!permanent!dipole!moment!and!a!change!in!molecular!cavity!size.!These! changes!elicit!the!polar!and!nonpolar!parts!of!the!dynamic!solvation!response,! respectively.79,111R117!There!are!a!small!set!of!probe!molecules!that!do!not!exhibit!a! change!in!permanent!dipole!moment,!however,!and!these!molecules!can!be! exploited!to!characterize!the!nonpolar!solvation!response!separately.!! -O S 3 SO3- N+ A O N H N HO NH2 O C B ! Figure!3.1!Structures!of!(a)!Cy5–lysine!adduct;!(b)!B3LYP/6‑31G(d)!structure!for! the!ground‑state!Cy5!chromophore;!(c)!horse‑heart!ferricytochrome!c (1HRC.pdb).11!The!protein!is!shown!in!a!ribbon!representation;!the!heme,!amino! acid!residues!Met80,!His18,!Cys14,!Cys17,!and!the!side!chains!of!the!lysine!residues! are!shown!in!a!stick!representation.!The!polypeptide!is!color!coded!from!red!to!blue! following!the!scheme!of!Englander!and!coworkers:57!residues!70–85!(red),!residues! 36–61!(yellow),!residues!20–35!and!60–70!(green),!and!the!N‑!and!C‑terminal!α! helices!(blue).! The!class!of!carbocyanine!dyes!that!includes!the!probe!called!Cy5!are!nonpolar! solvation!probes.!The!electronic!structure!of!the!Cy5!chromophore!is!comparable!to! ! 59! that!of!the!structurally!related!dyes!IR125!and!HDITCP,!which!have!been!discussed! by!Jonas!and!co‑workers.118!Two!resonance!structures!like!the!one!shown!in! Figure!3.1a,!each!with!a!formal!positive!charge!on!one!of!the!indole!nitrogen!atoms! at!the!end!of!the!conjugated!polymethine!chain!and!a!mirror!image!of!the!other,! contribute!equally!to!the!average!structure!of!Cy5.!The!π‑electron!density!and! charge!are!extensively!delocalized!over!the!full!length!of!the!conjugated!region;!the! * !π → π !transition!results!in!a!symmetrical!change!in!electron!density!with!respect! to!the!mirror!plane,!so!the!change!in!dipole!moment!is!negligible.!Thus,!the! time‑integrated!shift!to!the!red!of!the!continuous‑wave!fluorescence!spectrum!with! respect!to!the!absorption!spectrum!arises!in!Cy5!primarily!from!a!nonpolar! mechanism.! In!the!following,!we!describe!a!series!of!femtosecond!pump–continuum‑probe! experiments!employing!Cy5!as!a!probe!of!the!hydration!shell!of!ZnCytc.!Cy5!was! obtained!with!a!~10!Å!tether!terminated!with!a!succinimidyl!ester!group! (NHS‑ester)!to!allow!conjugation!with!surface!lysine!residues.!Using!the!continuum! viscoelastic!theory!advanced!by!Berg,79!the!shift!is!treated!in!terms!of!the!change!in! solute!size!or!shape!that!accompanies!the!optical!excitation!and!the!phonon‑induced! and!structural!responses!of!the!surrounding!solvent!cavity.!The!results!show! explicitly!that!the!hydration!shell!is!more!viscous!than!is!bulk!water!owing!to!the! observation!of!much!slower!dynamic!solvation!and!slower!out‑of‑plane!vibrational! dynamics.! ! 60! 3.2!Experimental!Section! 3.2.1!Sample!Preparation.!Cy5!solutions!in!water!were!prepared!using!the! amine!monoreactive!NHS!ester!of!Cy5!(GE!Healthcare,!PA25001,!see!Figure!3.1a)!in! a!25!mM!sodium!phosphate!buffer!solution!at!pH!7.0;!the!NHS!ester!hydrolyzes!to! yield!a!carboxylate!at!the!end!of!the!tether!after!prolonged!exposure!in!aqueous! solution.!! ZnII‑substituted!cytochrome!c!(ZnCytc)!was!prepared!from!horse‑heart! ferricytochrome!c using!the!procedure!described!in!Chapter!2.!The!Cy5–ZnCytc! complex!was!prepared!by!reacting!a!thawed!sample!of!ZnCytc!with!amine! monoreactive!N‑hydroxysuccinimidyl!(NHS)!ester!of!Cy5!to!prepare!nonspecifically! a!singly!labeled,!lysine!adduct!according!to!the!vendor's!protocol.!Figure!3.1c!shows! the!lysine!side!chains!of!ZnCytc!to!which!the!dye!could!bind.!The!reaction!was! conducted!at!a!protein!concentration!of!~3!mg/mL!at!room!temperature!in!a! 100!mM!sodium!bicarbonate!sample!buffer!solution!at!pH!9.3!with!enough!Cy5!dye! to!obtain!at!best!a!1:1!stoichiometry;!the!low!protein!concentration!favors!a!lower! adduct!yield.!Repeated!ultrafiltration!cycles!with!additions!of!25!mM!sodium! phosphate!buffer!solution!at!pH!7.0!over!YM10!membranes!(Millipore/Amicon)! were!used!to!separate!unreacted!and!weakly!bound!Cy5!from!the!protein!adducts.! The!adducts!were!concentrated!further!to!obtain!solutions!for!storage.!The!final! concentrated!product!was!stored!at!−80!°C!in!25!mM!sodium!phosphate!buffer! solution!at!pH!7.0.! For!use!in!the!femtosecond!pump–probe!experiments,!a!solution!of!Cy5!or! Cy5‑ZnCytc!were!prepared!by!diluting!concentrated!solutions!immediately!prior!to! ! 61! the!experiment!to!obtain!an!absorbance!of!0.3!for!a!path!length!of!1.0!mm!at!the! center!of!the!laser!spectrum!at!665!nm.!The!samples!were!held!in!the!femtosecond! pump–probe!spectrometer!at!room!temperature!(23!°C)!in!a!stirred!fused‑silica! cuvette!(1.0!mm!path!length).!The!sample's!absorption!spectrum!was!monitored!for! changes!arising!from!photochemistry!or!permanent!photobleaching.! 3.2.2!Mass!Spectrometry.!For!use!in!mass!spectrometric!analyses,!Cy5–ZnCytc! and!ZnCytc!were!treated!with!trypsin!to!obtain!peptide!fragments.!After!incubation! overnight,!the!samples!were!analyzed!using!a!Shimadzu!Axima!CFR!Plus!matrix– assisted!laser!desorption/ionization!time‑of‑flight!mass!spectrometer! (MALDI-TOF!MS).!The!samples!were!prepared!by!placing!1! µ L!of!sample!(33.2! µ M! ZnCytc!or!29.5! µ M!Cy5–ZnCytc!in!1!mM!phosphate!buffer)!on!a!MALDI!plate!with! 3! µ L!of!the!matrix! α ‑cyano‑4‑hydroxycinnamic!acid!in!3:1!acetonitrile:0.1%! trifluoroacetic!acid.!The!samples!were!allowed!to!dry!before!being!placed!into!the! mass!spectrometer.!For!mass!calibration,!the!YAGFLR!peptide!(726.4!Da),! bradykinin!(1059.6!Da),!and!angiotensin!I!(1297.5!Da)!were!used!as!standards.! Peaks!were!processed!using!the!mass!list!display!in!the!Shimadzu!Biotech! MALDI-MS!program.!! 3.2.3!Continuous‑wave!Absorption!and!Fluorescence!Spectroscopy.! Absorption!spectra!were!acquired!with!a!Hitachi!U‑4001!spectrophotometer!(2!nm! bandpass).!Fluorescence!spectra!were!obtained!using!a!home‑built!fluorescence! spectrometer!consisting!of!a!Jobin‑Yvon!AH10!100!W!tungsten‑halogen!light!source,! a!Jobin‑Yvon!H10!excitation!monochromator!(4!nm!bandpass),!an!Acton!Research! SP‑150!emission!spectrograph!(2!nm!bandpass),!and!a!Jobin‑Yvon!Symphony!CCD! ! 62! detector.!The!spatially!integrated!power!of!the!excitation!beam,!as!estimated!using!a! Coherent!Fieldmaster!power!meter!and!associated!silicon!photodiode!detector,!was! 10! µ W!at!522.9!nm.!Fluorescence!emission!spectra!were!acquired!as!the!average!of! twenty!60‑second!exposures!of!the!CCD!detector.!The!CCD!detector!employs!a!liquid! nitrogen!cooled,!back‑illuminated,!2000!⨉!800!pixel!silicon!detector!chip!(EEV! corporation).!A!300!groove/mm!diffraction!grating!(500!nm!blaze!wavelength)!was! mounted!in!the!emission!spectrograph,!resulting!in!a!270!nm!spectral!range!imaged! over!2000!vertically‑binned!channels!on!the!CCD!detector!chip.!As!presented!as!a! function!of!wavenumber,!the!fluorescence!intensities!are!multiplied!by!the!square!of! the!wavelength!in!order!to!compensate!for!the!fixed!(in!wavelength!units)!spectral! bandpass!of!the!emission!spectrograph.!The!absorption!instrument!is!controlled!by! Hitachi!UV!Solutions.!The!fluorescence!instrument!is!controlled!by!LabVIEW! (National!Instruments)!programs.! 3.2.4!Femtosecond!Spectroscopy.!Femtosecond!pump–continuum‑probe! experiments!were!conducted!with!pump!pulses!from!an!optical!parametric! amplifier!(OPA,!Coherent!OPA!9450),!which!was!driven!by!a!250‑kHz!regeneratively! amplified!Ti:sapphire!laser!(Coherent!RegA!9050!amplifier!and!Coherent!Mira‑Seed! oscillator).!The!oscillator!group‑delay!and!amplifier!were!pumped!continuously!by! Coherent!Verdi!V5!and!V10!Nd:YVO4!lasers.!The!signal‑beam!output!of!the!OPA!was! compensated!for!group‑delay!dispersion!by!an!SF10!Brewster!prism!pair!and! variably!delayed!by!a!retroreflector!on!an!optical!delay!line!driven!by!a!Melles‑Griot! Nanomover!actuator.!The!pump–pulse!duration!was!measured!to!be!45!fs!at!665!nm! by!a!zero‑background!autocorrelator!using!a!100‑μm!KDP!crystal;!the!spectrum!was! ! 63! determined!to!be!18!nm!in!width!(fwhm)!centered!at!665!nm!by!an!Ocean!Optics! U2000!spectrometer!(0.5!nm!bandpass).!! Probe!pulses!were!obtained!from!a!chirp‑compensated,!single‑filament! femtosecond!continuum,!which!was!generated!as!the!seed!pulses!for!the!first!gain! pass!in!the!OPA.!The!chirp!on!the!probe!beam!was!compensated!over!the! 500‑780!nm!range!using!a!pump‑delay!program!controlled!by!a!third‑order! polynomial!with!respect!to!the!probe!wavelength.!The!chirp!program!was! determined!using!optically!heterodyne‑detected!optical!Kerr‑effect!measurements! of!the!probe!arrival!delay!at!the!sample!position!as!a!function!of!probe!wavelength.! The!rise!time!(10%!to!90%)!for!the!pump–probe!signal!was!typically!75!fs!for!a! 4!nm!probe!bandpass.! For!pump–probe!measurements,!the!planes!of!linear!polarization!of!the!pump! and!probe!beams!were!set!to!be!54.7°!apart!as!incident!on!the!sample!using!calcite! polarizers!and!λ/2‑retarding!wave!plates.!The!probe!intensity!was!detected!by!a! Thorlabs!PDA‑55!amplified!photodiode!after!it!passed!through!the!sample!and!a! double‑subtractive!monochromator!(Spectral!Products!CM112)!with!a!4!nm! bandpass.!The!pump–probe!signal!was!obtained!as!the!normalized!pump‑induced! change!in!probe!transmission!(!ΔT /T )!signal!using!a!SRS!SR‑830!lock‑in!amplifier! referenced!to!the!sum!frequency!of!the!pump!and!probe!modulation!frequencies! (4.1!kHz!for!!ΔT ,!Palo!Alto!Research!model!300!chopper)!and!a!SRS!SR‑850!lock‑in! amplifier!referenced!to!the!probe!modulation!frequency!(2.5!kHz,!for!!T ).! 3.2.5!Computational!Chemistry.!The!optimized!structure!for!the!Cy5! chromophore!shown!in!Figure!3.1b!was!obtained!with!Gaussian!03119!using!the! ! 64! B3LYP!density!functional!and!the!6‑31G(d)!level!of!theory.!The!length!of!the!flexible! tether!between!the!tagged!lysine!residue's!α!carbon!and!the!N!atom!in!the!indole! moiety!at!the!other!end!of!the!tether!was!estimated!with!structures!optimized!using! the!UFF!molecular!mechanics!force!field.! 3.3!Results! 3.3.1!Mass!Spectrometry.2!In!order!to!assign!the!position!of!the!Cy5!molecule! on!the!surface!of!ZnCytc,!enzymatic!digestion!of!the!protein!was!followed!by! MALDI‑TOF!mass!spectrometry.!The!results!indicated!that!the!fragment!containing! peptides!81‑87!had!the!highest!probability!of!having!Cy5!conjugation,!which! indicates!conjugation!to!either!Lys86!or!Lys87.!The!fragment!containing!Lys22!was! the!next!most!likely!candidate,!followed!by!the!fragment!containing!Lys72.! According!to!Gibson!and!coworkers,120!the!six!most!reactive!lysines!of!the!18! lysines!in!cytochrome!c!are!residues!86,!25,!72,!13,!87,!and!22,!in!order!of! decreasing!reactivity.!With!our!results,!this!indicates!that!Lys86!is!the!most! probable!point!of!conjugation.!While!our!results!clearly!indicated!conjugation!to! three!peptide!fragments!(four!possible!lysines),!we!cannot!rule!out!the!possibility! that!conjugation!to!other!lysine!residues!occurs!on!occasion.!! 3.3.2!Continuous‑wave!Absorption!and!Fluorescence!Spectroscopy.! Figure!3.2!shows!the!room!temperature!absorbance!and!fluorescence!spectra!of! each!Cy5!in!water!with!the!femtosecond!pump!spectrum!overlaid.!The!spectra! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 2!The!mass!spectrometry!work!was!performed!by!Jerome!D.!Roscioli!of!the!Beck! Group.! ! 65! exhibit!a!main!band!(0–0!transition)!flanked!to!higher!and!lower!wavelengths,! respectively,!by!a!vibronic!satellite.!The!spacing!between!the!main!band!and!the! satellite!in!each!spectrum!is!around!1100–1200!cm−1,!so!the!excited!state!structure! is!displaced!mostly!likely!along!a!C‑C!or!C‑N!bond‑stretching!coordinate.!The! breadth!of!the!main!band!very!likely!conceals!unresolved!vibronic!structure!along! lower‑frequency!vibrational!modes.!The!approximate!mirror!symmetry!for!the! absorption!and!fluorescence!spectra!provides!strong!evidence!for!a!bound!excited! state!that!does!not!cross!to!a!product!state.!In!the!related!cyanine,! 1,1‑diethyl‑4,4‑cyanine!(1144‑C),!which!has!a!single!carbon!linker!between!the! terminal!ring!structures,!a!barrierless!(!E a < kBT )!cis–trans!isomerization!is!thought! to!occur!on!the!excited!state!potential!over!the!0.3!to!2!ps!timescale.121,122! Fluorescence!emission!during!the!crossing!of!the!barrier!between!the!cis!and!trans! configurations!gives!rise!to!a!pronounced!red!band,!making!the!absorption!and! fluorescence!spectra!distinctly!asymmetric.!The!mirror!symmetry!and!vibronic! progression!observed!for!Cy5!indicate!that!the!excited‑state!structure!is!displaced! along!in‑plane!and!possibly!out‑of‑plane!vibrational!coordinates,!but!it!is!unlikely! that!a!significant!fraction!of!the!excited‑state!population!executes!a!change!in! configuration.! ! 66! Relative Dipole Strength 1.0 0.5 0 12000 14000 16000 18000 -1 Frequency (cm ) 20000 ! Figure!3.2!Continuous‑wave!absorption!(blue)!and!fluorescence!(red)!spectra!of! Cy5!in!water!overlaid!with!the!laser!spectrum!(black)!and!normalized!to!maximum! peak!intensity.!! Relative Dipole Strength 1.0 0.5 0 12000 14000 16000 18000 -1 Frequency (cm ) 20000 ! Figure!3.3!Continuous‑wave!absorption!(blue)!and!fluorescence!(red)!spectra!of! Cy5–ZnCytc!overlaid!with!the!pump!spectrum!(black)!and!normalized!to!maximum! peak!intensity.!! ! 67! The!absorption!spectrum!for!the!Cy5–ZnCytc!adduct!(Figure!3.3)!exhibits!the! same!features!observed!in!the!spectrum!from!Cy5!in!water!in!addition!to!absorption! features!from!the!Q‑band!of!the!ZnII!porphyrin.!The!absorbance!peak!is!red!shifted! by!about!100!cm−1!relative!to!the!absorbance!peak!of!Cy5!in!water,!which!indicates! an!increased!polarizability!or!density!in!the!hydration!shell!compared!to!bulk!water.! The!peak!of!the!fluorescence!spectrum!of!Cy5–ZnCytc!is!red!shifted!40!cm−1!relative! to!the!peak!of!the!Cy5!fluorescence!spectrum,!which!is!an!indicator!of!the! reorganization!dynamics!for!fluorescence!reporting!slower!dynamic!solvation!in!the! hydration!shell.!In!spite!of!the!contribution!of!ZnCytc!to!the!absorbance!spectrum!of! the!Cy5–ZnCytc!adduct!and!the!shift!relative!to!the!spectra!of!Cy5!in!water,! excitation!by!the!laser!spectrum!is!expected!to!excite!only!Cy5,!not!the!ZnII! porphyrin,!so!the!resulting!fluorescence!spectrum!is!expected!to!be!a!result!of!Cy5! fluorescence,!not!ZnCytc!fluorescence.!Observed!changes!in!the!Cy5!fluorescence! spectrum!can,!therefore,!be!assigned!to!differences!in!the!immediate!environment!of! the!chromophore!which!will!be!further!elucidated!by!the!time‑resolved! spectroscopy.! The!solvation!reorganization!energy!observed!is!225!cm−1!for!Cy5!in!water!and! 195!cm−1!for!Cy5–ZnCytc.!Jonas!and!co‑workers118!found!the!solvation! reorganization!energy!for!HDITCP!and!IR125!to!be!270!cm−1!and!259!cm−1,! respectively.!These!dyes!were!found!to!have!no!change!in!solvent!reorganization! energy!as!the!polarity!of!the!solvent!was!varied.!The!conjugated!polyene!is!shorter! in!Cy5!than!IR125!or!HDITCP!by!one!single/double!bond!segment!but!is!otherwise! ! 68! isostructural,!so!a!similar!independence!for!the!solvation!reorganization!energy! with!respect!to!solvent!polarity!is!expected.! 3.3.3!Pump–Continuum!Probe!Spectroscopy.!Figure!3.4!shows!time‑resolved! pump‑probe!spectra!for!Cy5!in!water!with!probe!delays!from!−20!fs!to!54!ps.!The! change!in!transmittance!over!the!intensity!of!the!probe!at!that!wavelength!is! reported!as!!∆T /T .!Each!spectrum!shown!is!the!sum!of!photobleaching!(PB)!and! stimulated!emission!(SE)!signals,!which!are!positively!signed!reflecting!an!increase! in!transmittance,!and!an!excited‑state!absorption!(ESA)!signal,!which!is!negatively! signed!reflecting!a!decrease!in!transmittance.!The!early‑time!spectra!show!!PB+SE ! character!well!outside!of!the!hole‑burning!PB!region!(Figure!3.4!top!panels),! suggesting!that!the!homogeneous!line!width!is!broader!than!the!laser!spectrum's! bandwidth.!The!earliest‑time!spectrum!(Figure!3.4,!–20!fs)!shows!a!relatively! mirror‑symmetric!signal!around!the!0–0!vibronic!position!that!is!comparable!to!the! sum!of!the!absorption!and!fluorescence!dipole‑strength!spectra.!By!0!fs!the!blue! shoulder!is!decreasing!due!to!the!onset!of!an!ESA!signal!which!builds!and! blue‑shifts.!This!evolution!of!the!ESA!feature!is!mostly!complete!by!500!fs.!During! this!same!time!period,!the!red!shoulder!of!the!main!peak!grows!to!a!maximum!at! about!50!fs!owing!to!photobleaching!of!the!ground!state!and!red‑shifting!SE!due!to! solvation.!This!blue!shifting!ESA!and!red‑shifting!SE!during!this!time!period!is!also! consistent!with!excited‑state!twisting,!similar!to!what!Mathies,!Shank,!and! coworkers!observed!during!the!photoisomerization!of!rhodopsin123!and! bacteriorhodopsin.124!The!Cy5!molecule,!however,!does!not!appear!to!form!an! isomerization!product!as!evidenced!by!the!lack!of!a!red!tail!in!the!fluorescence! ! 69! spectra,!as!mentioned!above,!but!it!likely!experiences!excited‑state!motion!along! torsional!coordinates!due!to!the!loss!of! π ‑bonding!character!in!the!first!excited! state.!After!about!500!fs,!most!of!the!dynamic!character!of!the!spectra!is!complete,! and!the!spectra!decay!as!the!ground!state!is!repopulated.!These!results!indicate! contributions!from!excited‑state!motion!and!nonpolar!solvation.!! Figure!3.5!shows!a!similar!set!of!time‑resolved!pump‑probe!spectra!for! Cy5-ZnCytc.!The!time!evolution!of!the!spectra!is!comparable!to!that!for!Cy5!in!water,! but!there!is!more!line!broadening!and!the!spectra!change!much!more!slowly!with! time.!! In!both!sets!of!spectra,!the!pump‑probe!spectrum!evolves!over!time!as!the! excited!Cy5!molecule!moves!toward!the!minimum!of!the!excited!state!potential! energy!surface.!In!the!ground!state,!electron!density!is!distributed!across!the! terminal!ring!structures!of!Cy5!and!conjugated!polyene!connecting!them.!The! conjugation!is!disrupted!as!one!electron!is!excited!from!the!ground!state,!allowing! the!molecule!to!twist!as!it!moves!along!the!potential!energy!surface.!As!the!molecule! moves!in!the!excited!state!and!the!probe!pulse!arrives,!SE!or!ESA!evolve!and! eventually!decay!as!the!ground!state!is!repopulated.! ! 70! PB -20 fs 0 fs 20 fs ΔT/T 50 fs 100 fs 150 fs 540 fs 5 ps 54 ps 500 550 600 650 Wavelength (nm) 700 750 ! Figure!3.4.!Time‑resolved!pump‑probe!spectra!for!Cy5!in!water!with!probe!delays! from!−20!fs!to!54!ps.!! ! 71! PB -20 fs 0 fs 20 fs 100 fs ΔT/T 150 fs 500 fs 3 ps 24 ps 58 ps 500 550 600 650 Wavelength (nm) 700 750 ! Figure!3.5.!Time‑resolved!pump‑probe!spectra!for!Cy5–ZnCytc!with!probe!delays! from!−20!fs!to!58!ps.!! ! 72! Figures!3.6!and!3.7!show!two!time!scales!of!an!analysis!of!Cy5!in!water!obtained! by!calculating!the!mean!frequency!after!isolating!the!peaks!of!the!spectra!which! were!shown!in!Figure!3.4.!The!data!was!fit!to!the!equation:!! ! ! () ( ) f t = A1e −t/γ cos ω t − φ + A2e −t/τ 2 + A3e −t/τ 3 + A4e −t/τ 4 + A5e −t/τ 5 ! (3.1)! The!parameters!of!the!fit!are!shown!in!Table!3.1.!The!model!equation!is!used!to! describe!the!signal!in!order!to!compare!the!response!observed!in!bulk!water!and!the! hydration!shell.!The!plot!in!Figure!3.6!shows!an!initial!rapid!red!shift,!followed!by!a! blue!shift,!and!subsequent!oscillation!of!the!spectrum,!which!can!also!be!seen!at! longer!delays!in!Figure!3.7.!As!observed!in!Figure!3.4,!the!most!significant!changes! are!complete!by!500!fs,!which!is!related!to!the!shift!of!ESA.!The!oscillatory!response! comes!from!a!combination!of!the!oscillatory!motions!of!the!SE!and!ESA!parts!of!the! signal!given!that!the!window!over!which!the!mean!frequency!was!calculated!spans! only!the!central!PB‑SE!peak.!! () ν t = ! ! ∫ν1 dνν I (ν ,t ) ν2 ∫ν1 dν I (ν ,t ) ν2 ! (3.2)! Figures!3.8!and!3.9!show!similar!mean!frequency!analysis!for!Cy5–ZnCytc!on!the! same!time!scales!shown!in!Figures!3.6!and!3.7.!Note!that!the!overall!shift!of!the! mean!frequency!is!greater!for!Cy5–ZnCytc.!The!initial!rapid!red!shift!is!comparable! in!magnitude!to!that!observed!for!Cy5!in!water.!It!is!followed!by!a!blue!shift! approximately!double!that!seen!in!Cy5!in!water,!oscillation!that!is!not!as! pronounced,!and!a!final,!slow!blue!shift.!This!data!also!confirms!the!previous!results! that!the!ESA!shift!is!complete!by!500!fs.! ! ! ! 73! Table&3.1.&&Fit!parameters!for!the!mean!frequency!model!of!Cy5!in!water!and!Cy5‑ZnCytc,!as!shown!in!Figures!3.6@3.9.!!The! parameters!correspond!to!Equation!3.1.! & A! & !& !& A! & !! & A! & !! & A! & !! & A! & !! & Cy5-Water& 0.40& 58&fs& 136&cm−1& 0.17& 438&fs& -0.30& 758&fs& 0.13& 1.38&ps& ---& ---& Cy5-ZnCytc& 0.46& 77fs& 133&cm−1& -0.17& 287&fs& -0.01& 801&fs& -0.13& 4.01&ps& -0.22& 216&ps& & Table&3.2.&&Fit!parameters!for!the!720!nm!transients!modeled!to!fit!the!data!obtained!from!Cy5!in!water!and!Cy5@ZnCytc,!as! shown!in!Figures!3.10@3.13.!!The!parameters!correspond!to!Equation!3.3.! & A! & !! & A! & !! & A! & !! & A! & !! & A! & !! & Cy5-Water& 0.45& 23&fs& 0.25& 1.00&ps& -0.02& 3.85&ps& 0.28& 637&ps& ---& ---& Cy5-ZnCytc& 0.70& 13&fs& 0.13& 181&fs& 0.06& 2.20&ps& 0.03& 28.1&ps& 0.08& 890&ps& ! ! 74! -1 Mean Frequency (cm ) 15050 15000 0 1 2 3 4 5 Delay (ps) -1 Mean Frequency (cm ) ! Figure'3.6.'Time!evolution!of!the!mean!frequency!of!the!pump‑probe!spectrum!of! Cy5!in!water!at!short!delay!times.!The!data!points!are!overlaid!with!a!fit!to! Equation!3.1,!and!the!fit!parameters!are!listed!in!Table.!3.1.! 15050 15000 0 10 20 Delay (ps) 30 40 ! Figure'3.7.'Time!evolution!of!the!mean!frequency!of!the!pump‑probe!spectrum!of! Cy5!in!water.!The!data!points!are!overlaid!with!a!fit!to!Equation!3.1,!and!the!fit! parameters!are!listed!in!Table.!3.1.' ! 75! -1 Mean Frequency (cm ) 15000 14900 14800 0 1 2 3 4 5 Delay (ps) -1 Mean Frequency (cm ) ! Figure'3.8.'Time!evolution!of!the!mean!frequency!of!the!pump‑probe!spectrum!of! Cy5–ZnCytc!at!short!delay!times.!The!data!points!are!overlaid!with!a!fit!to! Equation!3.1,!and!the!fit!parameters!are!listed!in!Table.!3.1.! 15000 14900 14800 0 10 20 Delay (ps) 30 40 ! Figure'3.9.'Time!evolution!of!the!mean!frequency!of!the!pump‑probe!spectrum!of! Cy5–ZnCytc.!The!data!points!are!overlaid!with!a!fit!to!Equation!3.1,!and!the!fit! parameters!are!listed!in!Table.!3.1.' ! ! ! ! 76! The!720!nm!transient!in!Figures!3.10!and!3.11!shows!the!time!evolution!of!the! pure!SE!region,!fully!resolved!from!the!PB!(absorption)!spectrum.!The!data!shown! includes!a!data!set!at!short!times,!up!to!1!ps,!and!a!data!set!out!to!a!delay!of!150!ps.! The!transients!for!each!Cy5!in!water!and!Cy5–ZnCytc!are!modeled!by!a!sum!of! exponentials!convoluted!with!an!instrument!response!function:! ! ! ! () f x = A1e −t/τ 1 + A2e −t/τ 2 + A3e −t/τ 3 + A4e −t/τ 4 + A5e −t/τ 5 ! (3.3)! The!signal!for!Cy5!in!water!shows!an!instrument‑response!limited!rise,!reporting! exclusively!stimulated!emission!from!the!excited!state,!followed!by!a! multiexponential!decay!with!timescales!of!23!fs!and!1!ps!(see!Table!3.2).!Two! subsequent!timescales!of!3.9!and!637!ps!were!measured!on!the!same!sample!on!a! 150!ps!time!axis!(Figure!3.11).!Rapidly!damped!oscillations!arising!from!vibrational! coherence!are!clearly!visible!on!the!pump‑probe!transient!at!short!times!for!both! Cy5!in!water!and!Cy5–ZnCytc!(Figures!3.10!and!3.12).!The!timescales!observed!in! Cy5–ZnCytc!were!13!fs,!181!fs,!2.2!ps,!28!ps,!and!890!ps.!The!vibrational!coherence! is!not!visible!in!Figures!3.11!and!3.12!due!to!the!1!ps!step!size!used!for!this! transient.!Figures!3.12!and!3.13!show!the!oscillatory!components!of!the!residuals! (signal‑model)!from!Figures!3.10!and!3.11.!The!residuals!were!for!Cy5!in!water!and! Cy5–ZnCytc!were!plotted!in!Figures!3.14!and!3.15,!respectively.!! ! 77! ΔT / T −200 0 200 400 600 800 1000 Delay (fs) ΔT / T ! Figure'3.10.'Pump‑probe!transient!of!Cy5!in!water!with!the!probe!wavelength!at! 720!nm.!The!data!points!are!overlaid!with!a!fit!to!Equation!3.3;!the!fit!parameters! are!listed!in!Table!3.2.!The!delay!spacing!in!this!experiment!shows!vibrational! coherence!not!observed!with!greater!step‑size.! 0 20 40 60 80 Delay (ps) 100 120 140 ! Figure'3.11.'Pump‑probe!transient!of!Cy5!in!water!with!the!probe!wavelength!at! 720!nm.!The!data!points!are!overlaid!with!a!fit!to!Equation!3.3;!the!fit!parameters! are!listed!in!Table!3.2.!' ! 78! ΔT / T −200 0 200 400 600 800 1000 Delay (fs) ΔT / T ! Figure'3.12.'Pump‑probe!transient!of!Cy5–ZnCytc!with!the!probe!wavelength!at! 720!nm.!The!data!points!are!overlaid!with!a!fit!to!Equation!3.3;!the!fit!parameters! are!listed!in!Table!3.2.!The!delay!spacing!in!this!experiment!shows!vibrational! coherence!not!observed!with!greater!step‑size.' 0 20 40 60 80 Delay (ps) 100 120 140 ! Figure'3.13.'Pump‑probe!transient!of!Cy5–ZnCytc!with!the!probe!wavelength!at! 720!nm.!The!data!points!are!overlaid!with!a!fit!to!Equation!3.3;!the!fit!parameters! are!listed!in!Table!3.2.!! ! 79! 0.2 0 −0.2 0 100 200 300 400 500 600 Delay (fs) ! Figure'3.14.'Oscillatory!residuals!(data!−!fit)!from!the!pump‑probe!transient!of!Cy5! in!water!with!the!probe!wavelength!at!720!nm.!The!data!points!are!fit!to!a!set!of! damped!cosinusoids!using!a!LP‑SVD!program.!The!spectral!density!obtained!from! the!fit!is!shown!in!Figure!3.16.!! 0.2 0 −0.2 0 100 200 300 400 Delay (fs) 500 600 ! Figure'3.15.'Oscillatory!residuals!(data!−!fit)!from!the!pump‑probe!transient!of! Cy5–ZnCytc!with!the!probe!wavelength!at!720!nm.!The!data!points!are!fit!to!a!set!of! damped!cosinusoids!using!a!LP‑SVD!program.!The!spectral!density!obtained!from! the!fit!is!shown!in!Figure!3.17.!! ! 80! Figures!3.16!and!3.17!show!the!data!modeled!by!a!set!of!damped!cosinusoids! with!a!linear!prediction,!singular‑value‑decomposition!(LP–SVD)!program.125,126! The!model!includes!two!components!at!297!cm−1!and!431!cm–1!for!Cy5!in!water,! with!respective!normalized!amplitudes!of!0.2!and!0.8.!The!corresponding!model!for! the!Cy5–ZnCytc!transient!(Figure!3.17)!includes!modulation!components!at! 286!cm−1,!420!cm−1,!and!538!cm−1!with!respective!normalized!amplitudes!of!0.6,! 0.36,!and!0.04.!The!components!correspond!most!likely!to!out‑of‑plane!normal! modes!of!vibration!for!the!Cy5!chromophore.!These!vibrations!are!rapidly!damped! because!they!result!in!twisting!or!bending!of!the!conjugate!polyene!and!accordingly! effect!large‑amplitude!motions!of!the!indolium!end!moieties!against!the! Amplitude surrounding!solvent!cavity.! 0 100 200 300 400 500 600 700 800 -1 Frequency (cm ) ! Figure'3.16.!Spectral!density!obtained!from!the!LP‑SVD!fit!of!the!oscillatory!part!of! the!pump‑probe!transient!of!Cy5!in!water!with!the!probe!wavelength!at!720!nm!(see! Figure!3.14).! ! 81! Amplitude 0 100 200 300 400 500 600 700 800 -1 Frequency (cm ) ! Figure'3.17.'Spectral!density!obtained!from!the!LP‑SVD!fit!of!the!oscillatory!part!of! the!pump‑probe!transient!of!Cy5–ZnCytc!with!the!probe!wavelength!at!720!nm!(see! Figure!3.15).!Three!frequency!components!at!286!cm−1,!420!cm−1,!and!538!cm−1! were!found!for!Cy5–ZnCytc.!! 3.4'Discussion' The!design!of!this!study!helps!to!answer!the!question!of!whether!water!in!the! hydration!shell!is!more!viscous!than!bulk!water.!The!absorbance!and!fluorescence! spectra!demonstrate!displacement!of!the!Cy5!excited!state!structure,!but!not! transition!to!a!product!state.!The!pump‑probe!data!elucidates!the!mechanical! motion!of!Cy5.!The!mean!frequency!plot!for!each!Cy5–ZnCytc!and!Cy5!in!water! shows!damped!oscillation!due!to!the!diffusive!out‑of‑plane!motion!towards!the! excited!state!potential!minimum,!in!which!the!molecule!is!in!a!twisted!conformation.! The!SE!vibrational!coherence!is!damped!at!the!same!rate!in!bulk!water!and!in!the! hydration!shell,!which!indicates!in‑plane!modes!of!vibration.!The!overall!shift!to!the! blue!of!the!mean!frequency!is!a!result!of!the!ESA!shifting!to!the!blue.!This!is!a!direct! measure!of!dynamic!nonpolar!solvation!and!can!be!used!to!compare!the!relative! ! 82! timescales!in!bulk!water!and!the!hydration!shell,!which!was!confirmed!by!transient! grating!and!photon!echo!experiments.! The!mirror‑symmetry!and!vibronic!progression!of!the!fluorescence!and! absorbance!spectra!give!strong!evidence!that!the!excited!state!structure!is! displaced,!but!does!not!cross!to!a!product!state.!The!mirror!symmetry!of!the! absorbance!and!fluorescence!spectra!showed!that!even!as!the!excited!state!molecule! exhibits!damped!oscillation!of!a!twisting!motion,!Cy5!does!not!undergo!a!trans!to!cis! isomerization.!In!addition,!since!the!excited!Cy5!molecule!has!two!symmetric! resonance!structures!that!contribute!equally!to!the!structure,!we!conclude!that!the! dipole!moment!doesn't!change.!Structural!changes,!therefore,!whether!in‑plane!or! out‑of‑plane!vibrations,!are!the!primary!result!of!optical!excitation.!! The!pump‑probe!signals!give!information!on!the!mechanical!motion!of!the! chromophore!as!limited!by!the!surrounding!solvent,!whether!in!the!bulk!or!in!the! hydration!shell.!The!time!evolution!of!the!pump‑probe!signals,!both!in!Cy5!in!water! and!Cy5–ZnCytc!show!an!initial!evolution!consisting!of!predominantly!a!small!red! shift!of!the!stimulated!emission!portion,!which!should!overlap!with!the!fluorescence! and!with!the!motion!of!the!excited!state!absorption.!We!can!identify!if!this!occurs!by! observing!the!time!evolution!of!the!series!of!spectra.!At!early!time,!the!excited!state! absorption!spectrum!overlies!most!of!the!PB!spectral!region.!At!longer!time,!the!ESA! spectrum!shifts!to!the!blue.!As!it!shifts,!it!causes!the!region!of!the!PB!spectrum!to!act! like!it's!rising!due!to!the!fact!that!the!ESA!has!moved!to!the!blue!so!that!the!ESA! signal!is!no!longer!subtracted!from!the!positive!signal.!The!spectrum!also!involves! red‑shifting!of!the!stimulated!emission.!PB!is!stationary!by!definition,!except!for! ! 83! some!line!broadening,!which!is!due!to!the!IVR!and!spectral!diffusion.!The!motion!of! the!ESA!describes!twisting!of!the!chromophore!around!the!double!bonds,!between! the!two!cyanine!end!chromophores.!! One!of!the!things!we!can!measure!with!the!pump‑probe!signals!is!how!the! hydration!shell!limits!the!reorganizational!motions!of!that!chromophore.!One!way! we!can!measure!that!is!to!look!at!the!mean!frequency!by!integrating!over!the!central! peak.!This!primarily!tells!us!how!the!ESA!moves.!Bulk!water!clearly!exhibits!faster! dynamics!than!water!in!the!hydration!shell.!The!initial!red!shift!is!the!rapid!part!of! the!nonpolar!solvation!dynamics.79!In!water,!the!red!shift!is!followed!very!quickly! by!a!blue!shift!on!almost!the!same!timescale,!due!to!the!fast!part!of!the!ESA!motion! to!the!blue.!The!mean!frequency!then!oscillates!on!the!picosecond!timeframe,! finishing!with!a!red!shift!that!ends!around!10!ps!and!a!final,!very!small!blue!shift! that!extends!out!to!50!ps.!This!describes!a!damped!motion!of!the!chromophore!on! the!torsional!coordinates.!This!is!limited!to!some!degree!by!the!surrounding!solvent.! If!you!compare!the!mean!frequency!response!of!Cy5!in!water!to!Cy5‑ZnCytc,!the! oscillation!of!the!response!in!the!hydration!shell!of!ZnCytc!appears!more!damped,! though!the!magnitude!of!the!overall!response!is!larger.!In!addition!to!this!oscillation! being!more!damped!in!the!hydration!shell,!the!damping!time,! γ ,!of!the!oscillation!is! longer!in!the!hydration!shell.!This!is!one!measure!of!the!viscous!damping!of!Cy5! out‑of‑plane!motions.!The!magnitude!of!the!initial!red!shift!of!Cy5!is!comparable!in! both!environments.!The!next!blue!shift!is!significantly!larger!in!Cy5‑ZnCytc!and!then! the!oscillatory!behavior!is!damped,!even!on!an!absolute!scale.!Finally,!there!is!a!net! shift!of!the!signal!to!the!blue!for!Cy5–ZnCytc.!The!red!shift!seen!on!the!intermediate! ! 84! timescale!in!bulk!water!is!not!apparent!in!the!hydration!shell.!This!shows,!directly,! that!most!of!this!signal!response!is!due!to!the!motion!of!the!ESA!and!of!the! wavepacket!that!makes!the!stimulated!emission!move!back!and!forth.!When! comparing!the!longest!timescales,!!τ 4 !of!Cy5!in!water!and!!τ 5 !of!Cy5‑ZnCytc,!the! latter!is!160!times!greater,!indicating!a!significant!increase!in!viscosity.!The!longest! time!constant!in!the!Cy5–ZnCytc!response!(216!ps)!demonstrates!that!the!blue!shift! of!the!ESA!dominates!the!results!measured!by!mean!frequency,!which!occurs!as!the! system!moves!toward!the!excited!state!minimum!on!the!potential!energy!surface,! which!is!a!twisted!structure.!While!oscillations!are!damped!for!both!Cy5!in!water! and!Cy5–ZnCytc,!on!the!long!timescale,!we!can!clearly!see!that!the!response!on!the! hydration!shell!is!much!slower!than!that!in!the!bulk!water.!! Some!of!the!same!information!is!obtained!by!looking!at!the!stimulated!emission! directly!in!the!665nm!pump–720nm!probe!(Figures!3.10‑3.13).!This!probe! wavelength!primarily!gives!information!related!to!stimulated!emission.!The!fast!part! of!this!is!the!twisting!motion!along!vibrational!coordinates.!In!both!bulk!water!and! the!hydration!shell,!the!vibrational!coherence!damps!by!about!300!fs.!These!are! long‑range!out‑of‑plane!motions!that!are!initially!damped.!The!signal!then!responds,! going!to!completion!as!judged!by!the!mean!frequency,!and!is!mostly!complete!by!a! picosecond.!The!overall!population!decay!occurs!over!a!longer!timescale!in!water,! but!that!is!due!to!solvation!dynamics,!not!motion.!In!the!protein!case,!the!damping!of! the!signal!is!very!similar!or!perhaps!a!little!faster!damping!than!in!bulk!water,!but! then!the!blue!shift,!which!influences!the!mean!frequencies!shifting!to!the!blue!gives! rise!to!a!decay!in!the!SE,!and!that!is!much!slower!in!hydration!shell.!From!the! ! 85! pump‑probe!data,!we!conclude!that!the!slowest!part!of!the!response!measured!in! the!hydration!shell!is!at!least!on!the!30!ps!timescale!given!the!model.!The!long! component!is!due!to!mechanical!relaxation!of!the!chromophore!and!this!twisting! and!is!a!direct!measure!of!viscosity.!It!appears!that!the!timescale!in!the!hydration! shell!is!about!160!times!slower!for!the!bulk!water.!For!comparison!with!other! liquids,!the!increase!in!viscosity!in!the!hydration!shell!of!a!protein!indicates!a! viscosity!is!intermediate!between!the!viscosity!of!ethylene!glycol!and!glycerol.!The! viscosity!of!bulk!water!at!room!temperature!is!1.0!cP.!The!value!for!ethylene!glycol! is!16!cP,!and!for!glycerol!is!1400!cP.!With!an!approximately!160!times!increase!in! viscosity!compared!to!bulk!water,!we!can!estimate!about!160!cP,!which!is!ten!fold! that!of!ethylene!glycol!and!about!a!tenth!that!of!glycerol.!This!increased!viscosity! suggests!that!they!hydration!shell!contains!chains!of!water!that!are!longer!and/or! more!persistent!than!those!found!in!bulk!water.!This!occurs!because!the!presence!of! a!protein!interrupts!the!long‑range!hydrogen!bonding!structure!and!because!the! surface!of!the!protein!is!charged.!The!charges!on!the!surface!of!the!protein!are! expected!to!introduce!order!to!the!water!molecules,!whereby!they!assume!a! low‑energy!arrangement!along!the!protein!surface!of!an!ordered!hydrogen!bonding! network!that!can!extend!away!from!the!interface!and!persist!longer!temporally.!The! lysine!residues!are!primarily!on!the!front!side!of!the!protein,!where!the!overall! charge!is!positive!(see!Figure!3.1c),!which!may!impact!how!hydrogen!bonding! occurs!in!the!hydration!shell!and,!thereby,!impact!the!viscosity.!While!this!study!is! focused!on!the!hydration!shell!of!ZnCytc,!it!would!be!interesting!to!conduct!a!similar! study!using!a!protein!with!a!surface!containing!fewer!charged!amino!acids!to! ! 86! determine!how!the!number!of!surface!charges!or!whether!proximity!of!like!charges! impact!the!viscosity!of!the!hydration!shell.!! The!pump‑probe!signals!tell!us!mostly!about!mechanical!motion!that!is! frictionally!limited.!Our!results!show!two!significant!pieces!of!information:!the! mechanical/frictional!damping!on!the!fast!timescale!is!not!much!different!in!the! hydration!shell!than!bulk!water,!but!is!much!slower!in!the!hydration!shell!on!the! slow!timescale.! ! ! ! 87! CHAPTER'4' Conclusions' The!experiments!described!in!this!work!provide!an!improved!picture!for!the! nature!of!the!structural!changes!in!ZnCytc!upon!optical!excitation!and!give!further! insight!into!differences!between!the!behavior!of!water!in!the!hydration!shell!as! compared!to!the!bulk.!In!Chapter!2,!we!set!out!to!further!elucidate!the!nature!of!the! partially!unfolded!structures!produced!by!the!IVE!process!in!ZnCytc.!We!found!that! a!specific!omega!loop!of!the!protein!is!most!likely!to!be!involved!in!changing!the! environment!of!the!porphyrin.!In!Chapter!3,!we!set!out!to!describe!the!behavior!of! water!in!the!hydration!layer!by!probing!the!region!with!a!dye!attached!to!the!surface! of!the!protein.!We!found!that!the!viscosity!of!the!hydration!shell!is!much!greater! than!the!viscosity!of!water!in!the!bulk!because!the!water!molecules!are!polarized!by! surface!charges!and!probably!form!longer!or!more!persistent!hydrogen‑bonded! chains!than!in!bulk!water.!! In!Chapter!2,!we!reviewed!the!energy!landscape/protein!folding!funnel! hypothesis1`4,24,52,55,56!which!describes!the!potential!energy!surface!of!a!protein! as!the!protein!folds.!This!hypothesis!suggests!that!proteins!fold!(or!unfold)!through! a!range!of!trajectories!rather!than!a!single!discrete!pathway!of!intermediate!states.! The!IVE!experiment!was!initially!designed!as!a!method!to!unfold!the!protein,!ZnCytc,! using!optical!excitation.25,48!This!set!of!experiments!helped!elucidate!how!the! protein!is!affected!by!an!optically‑excited!intrinsic!ZnII‑porphyrin.!Our!results! indicate!that!an!omega!loop!(the!green!loop!in!the!Englander!color!scheme57)!of!the! ! 88! protein!undergoes!a!conformational!change!after!IVR!from!the!excited!porphyrin.! This!region!was!identified!to!be!adjacent!to!Cys14!and!Cys17,!which!are!connected! to!the!porphyrin!via!thioether!linkages.!Of!the!areas!adjacent!to!these!bonds,!the! identified!loop!was!chosen!based!on!how!its!movement!would!affect!the!porphyrin.! This!result!appears!to!be!dependent!upon!the!thioether!linkages!connecting!the! porphyrin!to!the!protein,!which!means!the!result!cannot!be!directly!applied!to! LHCII.!ZnCytc!was!originally!chosen!for!two!reasons:!it!contains!an!intrinsic! chromophore!and!is!a!model!for!a!single‑chlorophyll!containing!system.!! We!concluded!that!a!small!section!of!the!protein!adjacent!to!the!porphyrin!and! near!the!thioether!linkages!involving!Cys14!and!Cys17!undergoes!a!conformational! change!after!excess!vibrational!energy!is!transferred!via!IVR!from!the!ZnII! porphyrin.!We!reached!this!conclusion!because!the!IVE!profiles!obtained!at!multiple! temperatures!and!various!concentrations!of!Gdm+!all!show!a!red–blue–red!shift.! The!origin!of!the!transitions!in!the!IVE!profile!are!conformational!changes! (transition!I)!and!partial!unfolding!(transitions!II!and!III).!Further,!through‑bond! energy!transfer!is!rapid!relative!to!energy!transfer!by!collision,!so!IVR!is!most!likely! to!occur!through!the!thioether!linkages.!This!excess!energy!effects!change,! specifically!the!conformational!changes!and!partial!unfolding!associated!with!the! transitions!described.!Lastly,!the!loop!of!the!protein!adjacent!to!the!face!of!the! porphyrin!is!most!likely!to!affect!the! π ‑electron!density!of!the!porphyrin,!which!will! impact!fluorescence.!This!is!a!significant!result!because,!rather!than!a!specific! unfolding!trajectory,!it!indicates!that!a!range!of!partially!unfolded!structures!are! populated!during!spontaneous!protein!folding.!! ! 89! The!time‑integrated!experiment!presented!in!Chapter!2!would!not!be!well‑suited! for!use!with!a!multi‑chlorophyll!system!like!LHCII.!Due!to!the!presence!of!many! chromophores!in!LHCII,!the!position!of!the!initial!excitation!would!be!ambiguous! and!downhill!energy!transfer!would!result!in!vibrational!excitation!in!many! positions!in!the!protein!for!each!optical!excitation!event!that!would!occur.!In! addition,!the!chromophores!in!LHCII!are!not!bound!to!the!protein!by!thioether! linkages,!so!energy!transfer!that!occurs!will!not!take!place!through!bonds.!Energy! transfer!that!occurs!by!collision!is!expected!to!be!slower!than!through‑bond!energy! transfer.!The!vibrational!energy!is!likely!to!be!diffused!more!evenly!around!the! chromophore!than!a!system!where!energy!is!transferred!through!bonds.!! In!Chapter!3,!we!described!previous!work!studying!the!hydration!shell!of! proteins!and!the!conclusion!that!water!in!the!hydration!shell!behaves!differently! than!in!the!bulk.80`83,89`92!The!stability!of!a!native‑fold!protein!is!affected!by!the! hydration!shell!immediately!surrounding!the!protein,84`87!to!a!distance!at!least! 1.0!nm!from!the!surface.93!While!there!is!an!effect!on!water!when!it!is!in!the! presence!of!any!interface,96,97!the!effect!appears!more!pronounced!in!the!hydration! shell!of!a!protein!as!the!solvation!response!is!even!slower.6,48,98`103!Since!there!is! mechanical!coupling!of!the!protein!and!the!hydration!shell54,106,!this!experiment! was!designed!to!separate!the!results!shown!in!previous!work!from!what!would!be! observed!by!using!a!tethered!chromophore!in!the!hydration!shell.!The!hydration! shell!had!previously!been!modeled,82,83!so!we!set!out!to!test!those!results.!! The!experiment!was!designed!to!allow!description!of!the!hydration!shell!of!a! protein.!The!pump‑probe!experiment!elucidated!the!mechanical!motion!of!the! ! 90! cyanine!dye,!Cy5.!Specifically,!the!measured!timescales!in!bulk!water!and!the! hydration!shell!differed!significantly.!The!timescale!observed!in!the!hydration!shell! was!a!factor!of!160!greater!than!the!timescale!observed!for!Cy5!in!bulk!water.!The! fluorescence!and!absorbance!spectra!exhibit!mirror‑symmetry!and!vibronic! progression!indicating!that!the!excited!state!structure!is!displaced!relative!to!the! ground!state,!but!does!not!cross!to!a!product!state.!Even!as!the!molecule! experiences!twisting,!it!does!not!undergo!a!trans!to!cis!isomerization.!Since!the!Cy5! molecule!has!two!symmetric!resonance!structures!in!the!excited!state!that! contribute!equally!to!the!overall!structure,!it!is!clear!that!the!dipole!moment!does! not!change!when!the!molecule!is!excited.!This!indicates!that!optical!excitation! results!in!structural!changes,!whether!they!are!in‑plane!or!out‑of‑plane.!The! experiment!allows!a!measurement!of!how!the!hydration!shell!limits!reorganization! of!the!chromophore!molecule.!The!dynamics!in!the!hydration!shell!are!significantly! slower!than!those!seen!in!bulk!water.!In!the!hydration!shell!of!Cy5–ZnCytc,!the! magnitude!of!the!overall!response!is!larger!and!more!damped!than!that!of!Cy5!in! bulk!water.!In!addition!to!being!more!damped,!the!damping!time!is!longer!in!the! hydration!shell.!This!is!a!result!of!the!viscous!damping!of!Cy5!out‑of‑plane!motions.! While!there!is!damping!in!both!Cy5!in!bulk!water!and!Cy5!in!the!hydration!shell!of! ZnCytc,!the!response!observed!in!the!hydration!shell!is!much!slower!than!that!in! bulk!water.!! This!dissertation!contributes!to!understanding!how!energy!of!optical!excitation! is!distributed!by!vibrational!equilibration!in!chromoproteins,!such!as!those!involved! in!photosynthesis!or!in!vision,!prior!to!fluorescence!emission!and!further!elucidates! ! 91! the!behavior!of!water!in!the!hydration!shell!of!the!ZnCytc!system.!This!model!system! gives!information!that!may!be!useful!in!future!studies!on!LHCII!and!provides!a! building!block!for!further!research!within!the!hydration!shell!of!a!protein.!This! could!also!be!expanded!by!comparing!the!hydration!shell!of!a!protein!to!the!activity! near!the!surface!of!a!membrane‑bound!protein.!! ! ! ! ! ! ! ' 92! ! ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' APPENDIX' ! ! ' 93! APPENDIX' The!figures!presented!in!Appendix!A!are!a!supplement!to!Chapter!2.!Each!figure! is!presented!in!the!manner!shown!in!Figure!2.5a!representing!data!obtained!under! the!solution!conditions!listed.!Each!point!represents!the!0–0!peak!of!the! fluorescence!spectrum!obtained!at!2,000–10,000!cm−1!above!the!0–0!absorbance! peak,!(!ν IVE ) .!Each!plot!represents!the!evolution!of!the!continuous!wave! fluorescence!spectrum!as!a!function!of!!ν IVE ,!which!has!been!referred!to!as!an!IVE! profile.!The!data!points!of!each!plot!are!described!by!four!transitions.!Transitions!I,! II,!and!III!are!models!described!by!Equation!2.3.!Transition!IV!is!represented!by!a! smoothing!spline.!For!simplicity,!Figures!2.5!and!2.10!show!a!comparison!of!the! transitions!without!the!corresponding!data!points.!Figures!A1‑A11!include!all! temperatures!studied!and!have!the!same!range!to!allow!comparison!between!plots.! Figures!A12‑A21!include!all!Gdm+!concentrations!studied.!While!the!range!shown! for!the!Gdm+!plots!is!different!than!that!of!the!temperature!plots,!it!is!consistent!for! all!ten!Gdm+!figures.! ! 94! 17000 ν0-0 (cm-1) 16980 16960 16940 16920 2000 4000 6000 νIVE (cm-1) 8000 6000 νIVE (cm-1) 8000 10000 ! Figure'A1.'IVE!profile!of!ZnCytc!at!5!°C:!Evolution!of!the!wavenumber!of!the!peak! dipole!strength!in!the!0–0!peak!of!the!continuous‑wave!fluorescence!spectrum!from! ZnCytc!at!5!°C!as!a!function!of!!ν IVE ,!the!excitation!energy!above!the!wavenumber!of! the!0–0!absorption!peak.! 17000 ν0-0 (cm-1) 16980 16960 16940 16920 2000 4000 10000 ! Figure'A2.'IVE!profile!of!ZnCytc!at!10!°C:!Evolution!of!the!wavenumber!of!the!peak! dipole!strength!in!the!0–0!peak!of!the!continuous‑wave!fluorescence!spectrum!from! ZnCytc!at!10!°C!as!a!function!of!!ν IVE ,!the!excitation!energy!above!the!wavenumber! of!the!0–0!absorption!peak.! ! ! 95! 17000 ν0-0 (cm-1) 16980 16960 16940 16920 2000 4000 6000 νIVE (cm-1) 8000 6000 νIVE (cm-1) 8000 10000 ! Figure'A3.'IVE!profile!of!ZnCytc!at!20!°C:!Evolution!of!the!wavenumber!of!the!peak! dipole!strength!in!the!0–0!peak!of!the!continuous‑wave!fluorescence!spectrum!from! ZnCytc!at!20!°C!as!a!function!of!!ν IVE ,!the!excitation!energy!above!the!wavenumber! of!the!0–0!absorption!peak.! 17000 ν0-0 (cm-1) 16980 16960 16940 16920 2000 4000 10000 ! Figure'A4.'IVE!profile!of!ZnCytc!at!30!°C:!Evolution!of!the!wavenumber!of!the!peak! dipole!strength!in!the!0–0!peak!of!the!continuous‑wave!fluorescence!spectrum!from! ZnCytc!at!30!°C!as!a!function!of!!ν IVE ,!the!excitation!energy!above!the!wavenumber! of!the!0–0!absorption!peak.! ! 96! 17000 ν0-0 (cm-1) 16980 16960 16940 16920 2000 4000 6000 νIVE (cm-1) 8000 6000 νIVE (cm-1) 8000 10000 ! Figure'A5.'IVE!profile!of!ZnCytc!at!40!°C:!Evolution!of!the!wavenumber!of!the!peak! dipole!strength!in!the!0–0!peak!of!the!continuous‑wave!fluorescence!spectrum!from! ZnCytc!at!40!°C!as!a!function!of!!ν IVE ,!the!excitation!energy!above!the!wavenumber! of!the!0–0!absorption!peak.! 17000 ν0-0 (cm-1) 16980 16960 16940 16920 2000 4000 10000 ! Figure'A6.'IVE!profile!of!ZnCytc!at!50!°C:!Evolution!of!the!wavenumber!of!the!peak! dipole!strength!in!the!0–0!peak!of!the!continuous‑wave!fluorescence!spectrum!from! ZnCytc!at!50!°C!as!a!function!of!!ν IVE ,!the!excitation!energy!above!the!wavenumber! of!the!0–0!absorption!peak.! ! ! 97! 17000 ν0-0 (cm-1) 16980 16960 16940 16920 2000 4000 6000 νIVE (cm-1) 8000 6000 νIVE (cm-1) 8000 10000 ! Figure'A7.'IVE!profile!of!ZnCytc!at!60!°C:!Evolution!of!the!wavenumber!of!the!peak! dipole!strength!in!the!0–0!peak!of!the!continuous‑wave!fluorescence!spectrum!from! ZnCytc!at!60!°C!as!a!function!of!!ν IVE ,!the!excitation!energy!above!the!wavenumber! of!the!0–0!absorption!peak.! 17000 ν0-0 (cm-1) 16980 16960 16940 16920 2000 4000 10000 ! Figure'A8.'IVE!profile!of!ZnCytc!at!70!°C:!Evolution!of!the!wavenumber!of!the!peak! dipole!strength!in!the!0–0!peak!of!the!continuous‑wave!fluorescence!spectrum!from! ZnCytc!at!70!°C!as!a!function!of!!ν IVE ,!the!excitation!energy!above!the!wavenumber! of!the!0–0!absorption!peak.! ! 98! 17000 ν0-0 (cm-1) 16980 16960 16940 16920 2000 4000 6000 νIVE (cm-1) 8000 6000 νIVE (cm-1) 8000 10000 ! Figure'A9.'IVE!profile!of!ZnCytc!at!80!°C:!Evolution!of!the!wavenumber!of!the!peak! dipole!strength!in!the!0–0!peak!of!the!continuous‑wave!fluorescence!spectrum!from! ZnCytc!at!80!°C!as!a!function!of!!ν IVE ,!the!excitation!energy!above!the!wavenumber! of!the!0–0!absorption!peak.! 17000 ν0-0 (cm-1) 16980 16960 16940 16920 2000 4000 10000 ! Figure'A10.'IVE!profile!of!ZnCytc!at!85!°C:!Evolution!of!the!wavenumber!of!the!peak! dipole!strength!in!the!0–0!peak!of!the!continuous‑wave!fluorescence!spectrum!from! ZnCytc!at!85!°C!as!a!function!of!!ν IVE ,!the!excitation!energy!above!the!wavenumber! of!the!0–0!absorption!peak.! ! 99! 17000 ν0-0 (cm-1) 16980 16960 16940 16920 2000 4000 6000 νIVE (cm-1) 8000 6000 νIVE (cm-1) 8000 10000 ! Figure'A11.'IVE!profile!of!ZnCytc!at!90!°C:!Evolution!of!the!wavenumber!of!the!peak! dipole!strength!in!the!0–0!peak!of!the!continuous‑wave!fluorescence!spectrum!from! ZnCytc!at!90!°C!as!a!function!of!!ν IVE ,!the!excitation!energy!above!the!wavenumber! of!the!0–0!absorption!peak.! 17080 ν0-0 (cm-1) 17060 17040 17020 17000 16980 16960 2000 4000 10000 ! Figure'A12.'IVE!profile!of!ZnCytc!in!the!presence!of!0.0!M!Gdm+:!Evolution!of!the! wavenumber!of!the!peak!dipole!strength!in!the!0–0!peak!of!the!continuous‑wave! fluorescence!spectrum!from!ZnCytc!at!20!°C!as!a!function!of!!ν IVE ,!the!excitation! energy!above!the!wavenumber!of!the!0–0!absorption!peak.! ! ! 100! -1 ν0-0 (cm ) 17070 17040 17010 16980 16950 4000 6000 8000 10000 -1 νIVE (cm ) ! Figure'A13.'IVE!profile!of!ZnCytc!in!the!presence!of!0.25!M!Gdm+:!Evolution!of!the! wavenumber!of!the!peak!dipole!strength!in!the!0–0!peak!of!the!continuous‑wave! fluorescence!spectrum!from!ZnCytc!at!20!°C!as!a!function!of!!ν IVE ,!the!excitation! energy!above!the!wavenumber!of!the!0–0!absorption!peak.! -1 ν0-0 (cm ) 17070 17040 17010 16980 16950 4000 6000 8000 10000 -1 νIVE (cm ) ! Figure'A14.'IVE!profile!of!ZnCytc!in!the!presence!of!0.5!M!Gdm+:!Evolution!of!the! wavenumber!of!the!peak!dipole!strength!in!the!0–0!peak!of!the!continuous‑wave! fluorescence!spectrum!from!ZnCytc!at!20!°C!as!a!function!of!!ν IVE ,!the!excitation! energy!above!the!wavenumber!of!the!0–0!absorption!peak.! ! ! 101! -1 ν0-0 (cm ) 17070 17040 17010 16980 16950 4000 6000 8000 10000 -1 νIVE (cm ) ! Figure'A15.'IVE!profile!of!ZnCytc!in!the!presence!of!1.0!M!Gdm+:!Evolution!of!the! wavenumber!of!the!peak!dipole!strength!in!the!0–0!peak!of!the!continuous‑wave! fluorescence!spectrum!from!ZnCytc!at!20!°C!as!a!function!of!!ν IVE ,!the!excitation! energy!above!the!wavenumber!of!the!0–0!absorption!peak.! -1 ν0-0 (cm ) 17070 17040 17010 16980 16950 4000 6000 8000 10000 -1 νIVE (cm ) ! Figure'A16.'IVE!profile!of!ZnCytc!in!the!presence!of!1.5!M!Gdm+:!Evolution!of!the! wavenumber!of!the!peak!dipole!strength!in!the!0–0!peak!of!the!continuous‑wave! fluorescence!spectrum!from!ZnCytc!at!20!°C!as!a!function!of!!ν IVE ,!the!excitation! energy!above!the!wavenumber!of!the!0–0!absorption!peak.! ! ! 102! -1 ν0-0 (cm ) 17070 17040 17010 16980 16950 4000 6000 8000 10000 -1 νIVE (cm ) ! Figure'A17.'IVE!profile!of!ZnCytc!in!the!presence!of!2.0!M!Gdm+:!Evolution!of!the! wavenumber!of!the!peak!dipole!strength!in!the!0–0!peak!of!the!continuous‑wave! fluorescence!spectrum!from!ZnCytc!at!20!°C!as!a!function!of!!ν IVE ,!the!excitation! energy!above!the!wavenumber!of!the!0–0!absorption!peak.! -1 ν0-0 (cm ) 17070 17040 17010 16980 16950 4000 6000 8000 10000 -1 νIVE (cm ) ! Figure'A18.'IVE!profile!of!ZnCytc!in!the!presence!of!2.5!M!Gdm+:!Evolution!of!the! wavenumber!of!the!peak!dipole!strength!in!the!0–0!peak!of!the!continuous‑wave! fluorescence!spectrum!from!ZnCytc!at!20!°C!as!a!function!of!!ν IVE ,!the!excitation! energy!above!the!wavenumber!of!the!0–0!absorption!peak.! ! ! 103! -1 ν0-0 (cm ) 17070 17040 17010 16980 16950 4000 6000 8000 10000 -1 νIVE (cm ) ! Figure'A19.'IVE!profile!of!ZnCytc!in!the!presence!of!3.0!M!Gdm+:!Evolution!of!the! wavenumber!of!the!peak!dipole!strength!in!the!0–0!peak!of!the!continuous‑wave! fluorescence!spectrum!from!ZnCytc!at!20!°C!as!a!function!of!!ν IVE ,!the!excitation! energy!above!the!wavenumber!of!the!0–0!absorption!peak.! -1 ν0-0 (cm ) 17070 17040 17010 16980 16950 4000 6000 8000 10000 -1 νIVE (cm ) ! Figure'A20.'IVE!profile!of!ZnCytc!in!the!presence!of!4.0!M!Gdm+:!Evolution!of!the! wavenumber!of!the!peak!dipole!strength!in!the!0–0!peak!of!the!continuous‑wave! fluorescence!spectrum!from!ZnCytc!at!20!°C!as!a!function!of!!ν IVE ,!the!excitation! energy!above!the!wavenumber!of!the!0–0!absorption!peak.! ! ! 104! -1 ν0-0 (cm ) 17070 17040 17010 16980 16950 4000 6000 8000 10000 -1 νIVE (cm ) ! Figure'A21.'IVE!profile!of!ZnCytc!in!the!presence!of!5.0!M!Gdm+:!Evolution!of!the! wavenumber!of!the!peak!dipole!strength!in!the!0–0!peak!of!the!continuous‑wave! fluorescence!spectrum!from!ZnCytc!at!20!°C!as!a!function!of!!ν IVE ,!the!excitation! energy!above!the!wavenumber!of!the!0–0!absorption!peak.! ! ! ! ' 105! ' ' ! ! ! ! ! ! ! ! ! LITERATURE'CITED' ! ! ' 106! LITERATURE'CITED' ! (1)! Frauenfelder,!H.;!Sligar,!S.!G.;!Wolynes,!P.!G.! The!energy!landscapes!and! motions!of!proteins.! Science!1991,!254,!1598.! DOI:!10.1126/science.1749933.! (2)! Onuchic,!J.!N.;!Luthey`Schulten,!Z.;!Wolynes,!P.!G.! 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