The proposed research seeks to understand the energetics and design of b-sheet structure. Dr. Regan has successfully developed a model system in which to study b-sheet formation, based on a variant of the b1 domain of IgG-binding protein G (b1). b1 is a structurally and thermodynamically well characterized 56 residue protein. She can readily manipulate the gene that encodes it and purify large quantities of protein. In preliminary studies, by making a series of substitutions at solvent exposed positions, she has determined a thermodynamic scale for the b-sheet-forming propensities of the amino acids. Building on these findings, she also measured the energetics of pair-wise interactions between amino acids across two strands of an anti-parallel b-sheet at an H-bonded site. Her experimental results show strong correlations with statistically derived probabilities. In the current proposal, she goes forward to perform two additional pair-wise studies: a non-H-bonded anti-parallel site and an H-bonded parallel site. The statistically observed pairings at such sites are significantly different from those of her original study. She will follow up on this work by using NMR methods to structurally characterize the nature of the stabilizing interactions. Finally, she will pursue novel designs to address specificity versus stability in protein structure, by converting b-sheet into a-helix and vice versa. The first such design has been realized: a protein that has 50 percent identity to b1, but which adopts a helical conformation. Further such designs and their structural and thermodynamic characterization are proposed. The results of her studies will significantly enhance fundamental understanding of the energetics and design of b-sheet structure and lay the ground rules for the rational redesign of b-sheets.
