We are investigating the energetics and physical-chemical basis of the interactions that stabilize proteins. These studies include analyses of the stability of small, monomeric 13-sheet proteins in solution and also the role that sidechain-sidechain interactions play in modulating the rate of protein folding both in vivo and in vitro. In addition, our studies investigate the energetic balance and folding mechanism that determines whether a protein will be soluble or form amyloid fibrils in solution. We are also developing x-ray diffraction methods to gain high-resolution structural information on amyloid fibrils. The proteins we use for these studies are a series of variants of the B1 domain of IgG-binding protein G (beta1) and the Green Fluorescent Protein (GFP). Our studies with GFP have allowed us to design a novel system that has great utility for the detection of protein-protein interactions. We have dissected GFP into two halves that can only be reassembled if fused to two interacting proteins. We are using this system to investigate the affinity and specificity of protein-protein interactions. The specific example of tetratrcopeptide repeat (TPR) mediated protein-protein interactions is presented. The split GFP system also provides a means by which to identify binding partners for """"""""orphan proteins"""""""" whose interaction partners are not yet known.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM057265-08
Application #
7060739
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Basavappa, Ravi
Project Start
1998-05-01
Project End
2007-04-30
Budget Start
2006-05-01
Budget End
2007-04-30
Support Year
8
Fiscal Year
2006
Total Cost
$287,384
Indirect Cost
Name
Yale University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
043207562
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
Wang, Jimin; Gulich, Susanne; Bradford, Catharine et al. (2005) A twisted four-sheeted model for an amyloid fibril. Structure 13:1279-88
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
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
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