Understanding the mechanism of protein folding is one of the most important and challenging problems in biophysics. The ultimate goal is to bridge the information gap between amino acid sequence and three- dimensional structure. Our approach is to use iso-l and iso-2 cytochromes c as model systems to investigate how known changes in the primary structure affect the kinetic and equilibrium properties of protein folding reactions. The focus is on proteins with mutations at or near conserved sites. Our hypothesis is that structure, either pre-existing or rapidly formed, guides folding, and that understanding sequence-structure relationships for this structure is a key element in deciphering the folding code. We propose to characterize pre-existing or rapidly formed structure in several ways: 1) monoclonal antibodies will be used to measure the rate of formation and location of elements of protein surface with the topography of the native protein; 2) the efficiency of catalysis of folding by prolyl isomerases will probe the stability of structure near proline residues; 3) site directed mutant proteins arid double-jump kinetic assays will be used to determine if non native proline isomers or incorrect His-heme ligation traps rapidly formed structure; 4) scanning calorimetry will be used to measure mutation-induced changes in global stability; 5) thermodynamic cycles that combine equilibrium stability and electron binding will be used to detect residual structure in thermally unfolded proteins; 6) H-D exchange from folded proteins will be used to measure local stability, and to identify regions which unfold locally, globally, or which remain structured in the unfolded state; 7) H-D pulse labeling of folding intermediates will be used to determine the location and stability of rapidly formed structure; and through collaborations, 8) mutation-induced changes in the atomic resolution structure of folded proteins will be assessed by X-ray crystallography. These studies of folding of mutant proteins will provide an understanding of the mechanism by which early intramolecular interactions in folding influence the choice between alternative local structures, and will aid in deciphering the code relating amino acid sequence to tertiary structure and function.
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