Hydrogen bonds involving backbone C=O and NH groups constitute a large number of the native contacts in folded proteins. However, relatively little is known about the nature of their contribution to protein folding and stability. This is largely because backbone mutations in proteins are difficult to introduce by conventional site-directed mutagenesis protocols. Here we propose site-directed mutagenesis experiments utilizing total chemical synthesis strategies to study the role of backbone-backbone hydrogen bonds in the folding and stability of several model protein systems including: P22 Arc repressor, 4-oxalocrotonate tautomerase (40T), CopG, and protein L. Our experiments will involve the total chemical synthesis and the biophysical characterization of a series of different 40T, Arc repressor, CopG and protein L analogues that contain amide to ester bond mutations at specific locations in their polypeptide chains. The ester bond mutation is designed to modulate the hydrogen bonding characteristics of specific amide bonds in the polypeptide chains of these protein systems.The results of this work will be used to test five hypotheses about the fundamental role of backbone-backbone hydrogen bonds in protein folding reactions. We will determine: (1) if the stabilizing effects of all backbone-backbone hydrogen bonds in proteins are the same; (2) if the stabilizing effects of backbone-backbone hydrogen bonds located in similar regions (i.e. in the middle of an a-helix or at the end of a f3-sheet) of different protein structures are the same; (3) if the stabilizing effects of structurally equivalent backbone-backbone hydrogen bonds in protein's with the same backbone topology but different amino acid sequences (i.e. <25% sequence homology) are the same; (4) if the stabilizing effects of backbone-backbone hydrogen bonds protein folding intermediates are to those in the protein's native state; and (5) if backbone-backbone hydrogen bonds contribute to the stabilization of protein folding transition states.
