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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM057265-04
Application #
6386846
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Wehrle, Janna P
Project Start
1998-05-01
Project End
2003-04-30
Budget Start
2001-05-01
Budget End
2003-04-30
Support Year
4
Fiscal Year
2001
Total Cost
$143,111
Indirect Cost
Name
Yale University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Magliery, Thomas J; Regan, Lynne (2006) Reassembled GFP: detecting protein-protein interactions and protein expression patterns. Methods Biochem Anal 47:391-405
Magliery, Thomas J; Wilson, Christopher G M; Pan, Weilan et al. (2005) Detecting protein-protein interactions with a green fluorescent protein fragment reassembly trap: scope and mechanism. J Am Chem Soc 127:146-57
Wilson, Christopher G M; Kajander, Tommi; Regan, Lynne (2005) The crystal structure of NlpI. A prokaryotic tetratricopeptide repeat protein with a globular fold. FEBS J 272:166-79
Wang, Jimin; Gulich, Susanne; Bradford, Catharine et al. (2005) A twisted four-sheeted model for an amyloid fibril. Structure 13:1279-88
Goehlert, Virginia A; Krupinska, Ewa; Regan, Lynne et al. (2004) Analysis of side chain mobility among protein G B1 domain mutants with widely varying stabilities. Protein Sci 13:3322-30
Ramirez-Alvarado, Marina; Cocco, Melanie J; Regan, Lynne (2003) Mutations in the B1 domain of protein G that delay the onset of amyloid fibril formation in vitro. Protein Sci 12:567-76
Ramirez-Alvarado, Marina; Regan, Lynne (2002) Does the location of a mutation determine the ability to form amyloid fibrils? J Mol Biol 323:17-22
Stone, M J; Gupta, S; Snyder, N et al. (2001) Comparison of protein backbone entropy and beta-sheet stability: NMR-derived dynamics of protein G B1 domain mutants. J Am Chem Soc 123:185-6
Seewald, M J; Pichumani, K; Stowell, C et al. (2000) The role of backbone conformational heat capacity in protein stability: temperature dependent dynamics of the B1 domain of Streptococcal protein G. Protein Sci 9:1177-93