I. Equilibrium unfolding studies of cytochrome C. II. The role of helix 1 aspartates in the stability and conversion of prion protein

In order to improve methods to study protein structure and to obtain a detailed structural understanding of cytochrome c equilibrium unfolding, spectra have been obtained for horse heart cytochrome c at equilibrium in solutions of 0 to 7 M guanidine hydrochloride (GdnHCl) using absorption, fluorescence, circular dichroism (CD), and attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopies. Unfolding studies using ATR-FTIR spectroscopy were performed using deuterium substituted GdnHCl which enhanced the ability to measure the true protein IR spectrum in the amide I region where secondary structure can be deduced. Results of ATR-FTIR studies showed reductions in α-helix and increases in β-sheet at high denaturant concentrations, contrary to expectations of finding primarily random coil structure. Collectively, results of the various spectroscopic unfolding studies show that there are three regions of major conformational change. At low denaturant concentrations there is a slight decrease in the Fe-Met80 coordination, a loosening of α helices, and an increase in β-sheet structure. At mid-range GdnHCl concentrations, the changes include a significant decrease in α-helix, a complete loss of Fe-Met80 coordination, a dramatic increase in the Trp59-theme distance, and increases in β-sheet, random structure, and turns. At high denaturant concentrations there is a slight continuation of the increase in the Trp59-theme distance, a continued increase in β-sheet, and a decrease in random structure. These results suggest the unfolding of cytochrome c is not a two-state transition, but rather occurs through multiple structures with significant β-sheet structure in the denatured state.; In a separate study, the role of helix 1 aspartates in the stability and conversion of prion protein was investigated. Prion protein is involved in the pathogenesis of transmissible spongiform encephalopathy diseases, which include chronic wasting disease in deer and elk and Creutzfeldt-Jakob disease in humans. A key event in the pathogenesis of transmissible spongiform encephalopathies is the conversion of the normal α-helical prion protein, PrP-sen, to the abnormal, high β-sheet protease resistant form, PrP-res. It was proposed that the conversion mechanism involves critical interactions at helix 1 (residues 144–153) and that the helix is stabilized by intra-helix salt bridges between two aspartate-arginine ion pairs at positions 144 and 148 and 147 and 151, respectively. Three mutants designed to destabilize the helix 1 salt bridges by replacing the aspartates with either asparagines or alanines were compared to wild type PrP using CD spectroscopy and cell-free conversion reactions to assess differences in secondary structure stability and conversion efficiency. Thermal and chemical denaturation experiments indicated that the overall structures of the asparagine mutants are not substantially destabilized but they appear to unfold differently. Cell-free conversion reactions performed using conditions unfavorable to salt bridge formation showed no significant differences between conversion efficiencies of mutant and wild type proteins. Using conditions more favorable to salt bridge formation, the mutant proteins converted with up to four-fold higher efficiencies than the wild type protein. Thus, while spectroscopic data indicate that the salt bridges do not substantially stabilize PrP-sen, the cell-free conversion data suggest that D144, D147 and their respective salt bridges stabilize the molecule against the conversion of PrP-sen to PrP-res.