This proposal is directed toward a better understanding of the relationships between protein structure, stability and dynamics. In energy landscape terms we propose to investigate the landscape near its energy minimum, the nature of the energy barriers leading from the minimum to unfolded states, and the possible role of structured states seen under highly denaturing conditions in generating increased thermal stability. The proposal consists of 3 specific aims: (1) We propose to use modern nuclear magnetic resonance methods and especially relaxation dispersion techniques to detect and define minority conformations of proteins. These excited states can often be critical intermediates in ligand binding, folding-unfolding pathways and other events where structural change is important. Experiments are proposed to examine the role of core packing defects, low pH and denaturants on the nature and distribution of minority equilibrium species. (2) Develop the methods needed to examine the responses of single protein molecules to the application of mechanical forces designed to unfold the protein. This approach will attach a single protein molecule via two double-stranded DNA """"""""handles"""""""" to specific sites on two different beads. Force is exerted on the protein by pulling the beads apart using laser tweezers. This approach offers the ability to study protein folding by allowing direct measurement of the force needed to unfold a protein by pulling it apart from specific points. This will allow us to obtain a new perspective of the energy surface along a specific reaction coordinate corresponding to the distance between the points of attachment. (3) Understand the structural and the thermodynamic source of the thermal stability of proteins from hyperthermophilic organisms. Using the CheY protein from Thermotoga maritima as a model, we have found that its thermal stability is largely due to its unusually low change in heat capacity upon folding. This unusual heat capacity change seems to be the result of a highly structured unfolded state and experiments are proposed to investigate the structural bases of the heat capacity change and nature of the unfolded state.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM057766-09
Application #
6919291
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Basavappa, Ravi
Project Start
1998-08-01
Project End
2008-07-31
Budget Start
2005-08-01
Budget End
2008-07-31
Support Year
9
Fiscal Year
2005
Total Cost
$214,988
Indirect Cost
Name
University of California Santa Barbara
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
094878394
City
Santa Barbara
State
CA
Country
United States
Zip Code
93106
Barnes, Ryan; Sun, Sheng; Fichou, Yann et al. (2017) Spatially Heterogeneous Surface Water Diffusivity around Structured Protein Surfaces at Equilibrium. J Am Chem Soc 139:17890-17901
Kingston, Richard L; Hamel, Damon J; Gay, Leslie S et al. (2004) Structural basis for the attachment of a paramyxoviral polymerase to its template. Proc Natl Acad Sci U S A 101:8301-6
Eldridge, Aimee M; Kang, Hyun-Seo; Johnson, Eric et al. (2002) Effect of phosphorylation on the interdomain interaction of the response regulator, NarL. Biochemistry 41:15173-80
Griswold, Ian J; Dahlquist, Frederick W (2002) The dynamic behavior of CheW from Thermotoga maritima in solution, as determined by nuclear magnetic resonance: implications for potential protein-protein interaction sites. Biophys Chem 101-102:359-73
Mulder, Frans A A; Hon, Bin; Mittermaier, Anthony et al. (2002) Slow internal dynamics in proteins: application of NMR relaxation dispersion spectroscopy to methyl groups in a cavity mutant of T4 lysozyme. J Am Chem Soc 124:1443-51
Skrynnikov, N R; Mulder, F A; Hon, B et al. (2001) Probing slow time scale dynamics at methyl-containing side chains in proteins by relaxation dispersion NMR measurements: application to methionine residues in a cavity mutant of T4 lysozyme. J Am Chem Soc 123:4556-66
Mulder, F A; Skrynnikov, N R; Hon, B et al. (2001) Measurement of slow (micros-ms) time scale dynamics in protein side chains by (15)N relaxation dispersion NMR spectroscopy: application to Asn and Gln residues in a cavity mutant of T4 lysozyme. J Am Chem Soc 123:967-75
Goto, N K; Skrynnikov, N R; Dahlquist, F W et al. (2001) What is the average conformation of bacteriophage T4 lysozyme in solution? A domain orientation study using dipolar couplings measured by solution NMR. J Mol Biol 308:745-64
Yang, G; Cecconi, C; Baase, W A et al. (2000) Solid-state synthesis and mechanical unfolding of polymers of T4 lysozyme. Proc Natl Acad Sci U S A 97:139-44
Mulder, F A; Hon, B; Muhandiram, D R et al. (2000) Flexibility and ligand exchange in a buried cavity mutant of T4 lysozyme studied by multinuclear NMR. Biochemistry 39:12614-22

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