Dr. Daggett requests continuation support to perform computer simulations of protein denaturation in solution. Considerable progress with unfolding simulations during the last funding period is described, with respect to establishing the methods, devising ways to identify and test models of transition states, developing analysis tools for the evaluation of residual structure in partially unfolded and denatured proteins, developing approaches for comparison of simulation results with experiment, and predicting experimental outcomes. But, there are still a number of issues that need to be addressed. High temperature molecular dynamics simulations of the unfolding of small globular proteins will continue continue, but huge differences between the experimental time scale of protein unfolding and what is accessible with current computer power. The validity of this approach will be evaluated by performing simulations at a variety of temperatures, using chemical denaturants, and performing temperature-quenched simulations. Also, while it is desirable to have the ability to follow a single protein over time, now multiple simulations will be performed to ensure that the effects observed are representative of the much larger ensemble probed experimentally, and the transition state ensembles identified from the simulations tested rigorously, to ensure that they are reasonable. The project will be extended to free energy perturbation calculations to provide more direct comparisons with experimental results. Finally, mutant proteins with altered folding behavior are being designed, based solely on the molecular dynamics-generated models. The mutants are being produced and experimentally evaluated in the lab of a collaborator, Prof Alan Fersht, an eminent Cambridge UK enzymologist.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM050789-08
Application #
6525818
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Wehrle, Janna P
Project Start
1995-08-01
Project End
2004-07-31
Budget Start
2002-08-01
Budget End
2003-07-31
Support Year
8
Fiscal Year
2002
Total Cost
$253,657
Indirect Cost
Name
University of Washington
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195
Towse, Clare-Louise; Vymetal, Jiri; Vondrasek, Jiri et al. (2016) Insights into Unfolded Proteins from the Intrinsic ?/? Propensities of the AAXAA Host-Guest Series. Biophys J 110:348-361
Towse, Clare-Louise; Rysavy, Steven J; Vulovic, Ivan M et al. (2016) New Dynamic Rotamer Libraries: Data-Driven Analysis of Side-Chain Conformational Propensities. Structure 24:187-199
Childers, Matthew Carter; Towse, Clare-Louise; Daggett, Valerie (2016) The effect of chirality and steric hindrance on intrinsic backbone conformational propensities: tools for protein design. Protein Eng Des Sel 29:271-80
Rysavy, Steven J; Beck, David A C; Daggett, Valerie (2014) Dynameomics: data-driven methods and models for utilizing large-scale protein structure repositories for improving fragment-based loop prediction. Protein Sci 23:1584-95
Rysavy, Steven J; Bromley, Dennis; Daggett, Valerie (2014) DIVE: a graph-based visual-analytics framework for big data. IEEE Comput Graph Appl 34:26-37
Merkley, Eric D; Rysavy, Steven; Kahraman, Abdullah et al. (2014) Distance restraints from crosslinking mass spectrometry: mining a molecular dynamics simulation database to evaluate lysine-lysine distances. Protein Sci 23:747-59
Towse, Clare-Louise; Hopping, Gene; Vulovic, Ivan et al. (2014) Nature versus design: the conformational propensities of D-amino acids and the importance of side chain chirality. Protein Eng Des Sel 27:447-55
Bromley, Dennis; Rysavy, Steven J; Su, Robert et al. (2014) DIVE: a data intensive visualization engine. Bioinformatics 30:593-5
Bromley, Dennis; Anderson, Peter C; Daggett, Valerie (2013) Structural consequences of mutations to the *-tocopherol transfer protein associated with the neurodegenerative disease ataxia with vitamin E deficiency. Biochemistry 52:4264-73
Wang, Dan; McCully, Michelle E; Luo, Zhaoxiong et al. (2013) Structural and functional consequences of cardiac troponin C L57Q and I61Q Ca(2+)-desensitizing variants. Arch Biochem Biophys 535:68-75

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