The Human Immunodeficiency Virus (HIV) is an enveloped retrovirus responsible for causing the Acquired Immunodeficiency Syndrome (AIDS) in humans. The virus vesicle's membrane contains the gp120/gp41 protein expressed on the outside of the virus, which is responsible for recognizing and fusing the virus vesicle with the target cell, creating a fusion pore leading to infection of the target cell. Currently there are no high resolution structures for the gp41 protein's fusion peptide (FP) during the infection process. Solid state nuclear magnetic resonance (SSNMR) spectroscopy provides the ability to obtain high resolution structural information for proteins in their native environment.This dissertation project required the development of a practical methodology for creating isotopically labeled FP-Hairpin where the labels are located in the FP region of a gp41 protein construct. Synthetically made FP23(linker) containing isotopic 13C, 15N labels and recombinantly expressed Hairpin in E. coli bacteria were ligated together using the native chemical ligation (NCL) reaction to produce the 115 residue FP-Hairpin construct in ~20% yield and 2 weeks time. FP-Hairpin allows for the study of the FP domain of gp41 in a lipid membrane environment in the post fusion, low energy six helix bundle (SHB) formation. Using 2D SSNMR experiments with selectively placed isotopic labels in the FP region provided the ability to probe secondary and tertiary structure of the protein in a membrane environment. Major results of this project are (1) the development of methodology to produce the 115 residue FP-Hairpin with isotopic labels using the NCL reaction and (2) SSNMR studies of the FP-Hairpin in lipid membrane environments. FP-Hairpin sample preparation time was reduced from 2 months to 2 weeks and NCL efficiency was improved from ~4% to ~20% yields. SSNMR was used to probe the secondary and tertiary structure of the FP-Hairpin protein in lipid membrane environments by using 2D 13C-13C experiments at magnetic fields of 9.4 T and 21.1 T. Results from the SSNMR experiments showed that with the 21.1 T instrument and the E-free probe, 13C peak signal to noise per scan was 5-fold higher and line widths 2-fold narrower than that which was obtained at the 9.4 T instrument and non E-free probe. Time savings of at least 50% were obtained at 21.1 T compared to the 9.4 T instrument. Prior to this work, the 21.1 T magnet had not been utilized to any great extent by the Weliky group. Thus this project can be summed up as a proof of concept project which highlights sample preparation and SSNMR work that previously had not been attempted. By utilizing the SSNMR experiments it was determined that the FP-Hairpin construct can have both the helical and &beta-strand secondary structure in the FP region in the presence of cholesterol containing membranes. The &beta-strand conformation agrees with the previous work for FP peptides. However, the tertiary structure was not the same, as Alanine-6 and Glycine-10 were observed to have cross peaks in the FP23 peptide at long mixing times, which were absent in the FP-Hairpin construct, suggesting that the registry of the two constructs FP23 and FP-Hairpin are different.
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