Solid state nuclear magnetic resonance (SSNMR) spectroscopy provides the opportunity to obtain high resolution data regarding the chemical environment of NMR active nuclei in solid and semi-solid samples. Of particular interest for study by SSNMR are biological molecules like proteins, as NMR provides a way to determine properties of these molecules such as secondary structure, internuclear distances, and dynamics. My dissertation project consisted of several different applications of SSNMR to study biological systems, as well as the preparation of these systems for study. gp41 is a protein present on the surface of virions of the human immunodeficiency virus (HIV). The protein gp41 is a glycoprotein which aids in the process of viral entry into the human host T cells by catalyzing the process of membrane fusion between the viral membrane and the T cell plasma membrane. Due to its implication in this process, it has been an attractive target for anti-HIV drug development. I produced in E. coli and purified an ectodomain construct of the gp41 protein called Fgp41 which included the catalytic fusion peptide. Structural analyses by circular dichroism spectroscopy and rotational echo double resonance (REDOR) SSNMR indicated that the protein was folded into the post-fusion low energy six helix bundle conformation. This was further supported by functional assays that showed little lipid-mixing ability of the protein. REDOR SSNMR was used to obtain high resolution structural information about the protein while associated with lipid membranes. This is the first example of atomic resolution structural data of the fusion peptide embedded into lipid membranes in the context of the protein. Human proinsulin is the biological precursor to the insulin hormone, which has therapeutic effects for people with the metabolic disease diabetes mellitus. Synthetic insulin is produced in many ways, including through recombinant protein expression in E. coli as the precursor protein proinsulin. It is documented that proinsulin is sequestered within inclusion bodies after recombinant expression, and drastic measures are taken to denature and refold the protein to produce bioactive insulin. By utilizing SSNMR, the REDOR pulse sequence, and selective isotopic labeling schemes, I was able to probe the secondary structure of human proinsulin within bacterial inclusion bodies. Both helical and β-strand conformations of the protein were observed in the A and B chains, while C chain (which is cleaved during the processing to form insulin) exhibited primarily neither helical nor β-strand chemical shifts. Recombinant expression in E. coli is a major way of producing protein for structural and functional studies. Different proteins express to different levels within E. coli, and for proteins that are difficult to solubilize, it is often difficult to determine whether they are expressing at all. By utilizing SSNMR, REDOR, and isotopically labeled whole E. coli cells I was able to detect the level of recombinant protein expressed. The NMR spectrum is simplified if the sample preparation includes a step to remove all soluble proteins. By comparison to a standard curve, I was able to determine the level of recombinant protein expression in mg protein produced per liter of bacterial cell culture for several different protein constructs. This is the first method of recombinant protein expression quantification in whole cells or insoluble cell pellets.
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