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 #
5R01GM057766-02
Application #
6019430
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1998-08-01
Project End
2002-07-31
Budget Start
1999-08-01
Budget End
2000-07-31
Support Year
2
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Oregon
Department
Type
Organized Research Units
DUNS #
948117312
City
Eugene
State
OR
Country
United States
Zip Code
97403
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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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