The experiments outlined in this proposal are aimed at identifying and controlling the ensemble of conformations that comprise the native state of a protein. In order to appreciate how a protein's sequence encodes a folded, functional molecule, we need to understand much more than just its native structure. Encoded in the primary sequence is the entire energy landscape. Non-native regions of the landscape are important for directing the stability and folding of a protein, and modulations in this ensemble play a crucial role in misfolding, signaling, allostery, and turnover. A major hurdle in our ability to go from sequence to function is our lack of understanding of these non-native states. Many different sequences can encode the same three dimensional structure, yet their function and dynamics vary dramatically;small variations can have effects that range from undetectable to pathological. These differences are often a consequence of subtle changes in non-native regions of the landscape. Here, we propose experiments directed at determining and controlling different features of the landscape using a mix of new and established experimental techniques and systems. Specifically, the aims of this proposal are:
Specific aim 1 : Single molecule studies of protein conformational changes a. Explore the unusual mechanical properties of non-native states b. Obtain energetic information from these inherently non-equilibrium studies c. Determine the mechanical coupling in repeat proteins Specific aim 2: Examine perturbations of the free energy landscape. a. Explore the energy landscape of thermophiles b. Probe the energy landscape of RNase H using mutagenesis and MPAX:mis-incorporation proton alkyl exchange Specific Aim 3: The de novo design of function through modulation of conformational ensembles. a. Design of a controllable allosteric switch b. Demonstrate control by ligand binding and mechanical tension

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM050945-17
Application #
7846220
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Wehrle, Janna P
Project Start
1994-05-01
Project End
2011-09-14
Budget Start
2010-06-01
Budget End
2011-09-14
Support Year
17
Fiscal Year
2010
Total Cost
$301,120
Indirect Cost
Name
University of California Berkeley
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
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