This proposal is directed towards obtaining a better understanding of the relationships between protein structure, stability and dynamics using T4 lysozyme as a model system. The proposal presents four specific aims: 1) The folding pathway connecting the folded and unfolded states will be investigated, with specific emphasis on the structure and dynamics of an early kinetic intermediate and equilibrium states that appear related to it. Approaches are described to monitor the relative rates of encounter of various parts of the polypeptide chain using an excimer probe.; 2) The mechanism of coupling between different parts of the sequence, that fold as an apparent unit in the wild-type protein but become uncoupled in certain mutants. Pulsed hydrogen exchange methods will be employed to define the rates at which different parts of the protein attain native-like structure.; 3) The accessibility of water and small hydrophobic ligands to cavities in the core of T4 lysozyme will be examined.; 4) A major effort will be undertaken to develop the methods necessary to observe and interpret the effects of the application of mechanical forces designed to unfold small assemblies of proteins attached to the tip of an atomic force microscope. These types of measurements will provide a new perspective of the energy landscape of proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM057766-04
Application #
6386921
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Wehrle, Janna P
Project Start
1998-08-01
Project End
2002-07-31
Budget Start
2001-08-01
Budget End
2002-07-31
Support Year
4
Fiscal Year
2001
Total Cost
$211,948
Indirect Cost
Name
University of Oregon
Department
Type
Organized Research Units
DUNS #
948117312
City
Eugene
State
OR
Country
United States
Zip Code
97403
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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