Total chemical synthesis and biophysical characterization of backbone-modified protein analogues

Hydrogen bonding is undoubtedly a major force in protein folding and stability. However, the exact nature and magnitude of the contributions of backbone-backbone hydrogen bonding to protein folding reactions have been difficult to examine experimentally. The work presented here describes the synthesis and biophysical characterization of several backbone-modified analogues of Bacteriophage P22 Arc repressor and CopG. The protein analogues in this study were designed to facilitate a comparative analysis of the role of backbone-backbone hydrogen-bonding interactions in the folding and stability of these two model protein systems that share the same three-dimensional structure but no primary sequence homology.; Detailed here is the first total chemical synthesis of Arc repressor and CopG using highly optimized solid phase peptide synthesis (SPPS) protocols for Boc-chemistry. Both Arc repressor and CopG were effectively synthesized in a stepwise fashion to give reasonably good yields of pure material. The work in this dissertation is also an account of the progress that has been made with respect to the development of CopG as a model protein system for the study of protein folding reactions. The initial efforts to characterize the unfolding reaction of CopG have shown promising results for a two-state model of protein folding. CopG unfolds in a reversible manner and Guanidinium Chloride-induced equilibrium unfolding data for wild-type CopG, as well as a fluorescent analogue of the wild-type sequence, lend support that the CopG unfolding reaction might be best described using a two-state model of protein unfolding.; Together these two model protein systems, combined with total chemical synthesis were employed in a comparative study of selected backbone-backbone hydrogen bonding interactions in Arc repressor and CopG. Backbone amide-to-ester bond mutations were incorporated at seven different positions within both the first alpha-helices and the beta-sheet dimer interfaces in these model systems. The comparative analysis of the backbone-modified Arc repressor and CopG analogues made within these elements of secondary structure suggest that the deletion of structurally similar backbone-backbone hydrogen bonds in these proteins perturbs the stability and structure to different extents.