The long-term objective of this research program is to understand the relationship between the amino acid sequences of proteins and their three-dimensional structures and stabilities. This is one of the central, unsolved problems in molecular biology and is important because protein folding is a prerequisite of most biological processes. In addition, much of the promise of biotechnology for improving human health depends on our eventual ability to manipulate the function of a protein by altering its sequence. We will use P22 Arc repressor, a dimeric protein of known structure, to study the effects of sequence changes produced by directed or combinatorial mutagenesis on the structure, stability, and folding kinetics of the protein. Because its is small and accessible to genetic, biophysical, and structural study, the Arc system is one of the premier models for studying the sequence determinants of protein folding and association. In other studies, we will investigate the conformational determinants of the prion state, using the URE2 protein of yeast as a model system. Prions provide a fascinating but poorly understood phenomena in which an altered state of the protein, which arises rarely but spontaneously, can be stably propagated in a dominant, epigenetic fashion by a mechanism in which altered molecules cause normal molecules to assume the altered prion state. In all studies, thermodynamic stability will be investigated by reversible denaturation; changes in folding and unfolding kinetics will be determined by stopped-flow methods; and conformational properties of selected molecules will be probed by x-ray crystallography or NMR. The data acquired will be important for testing and refining our understanding of the sequence determinants of protein structure and stability and for evaluating algorithms that seek to predict the effects of sequence changes on protein structure and function.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI015706-21
Application #
2886375
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Beisel, Christopher E
Project Start
1979-05-01
Project End
2002-04-30
Budget Start
1999-05-01
Budget End
2000-04-30
Support Year
21
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
City
Cambridge
State
MA
Country
United States
Zip Code
02139
Olivares, Adrian O; Kotamarthi, Hema Chandra; Stein, Benjamin J et al. (2017) Effect of directional pulling on mechanical protein degradation by ATP-dependent proteolytic machines. Proc Natl Acad Sci U S A :
Sauer, Robert T (2013) Mutagenic dissection of the sequence determinants of protein folding, recognition, and machine function. Protein Sci 22:1675-87
Gur, Eyal; Vishkautzan, Marina; Sauer, Robert T (2012) Protein unfolding and degradation by the AAA+ Lon protease. Protein Sci 21:268-78
Glynn, Steven E; Nager, Andrew R; Baker, Tania A et al. (2012) Dynamic and static components power unfolding in topologically closed rings of a AAA+ proteolytic machine. Nat Struct Mol Biol 19:616-22
Baker, Tania A; Sauer, Robert T (2012) ClpXP, an ATP-powered unfolding and protein-degradation machine. Biochim Biophys Acta 1823:15-28
Román-Hernández, Giselle; Hou, Jennifer Y; Grant, Robert A et al. (2011) The ClpS adaptor mediates staged delivery of N-end rule substrates to the AAA+ ClpAP protease. Mol Cell 43:217-28
Aubin-Tam, Marie-Eve; Olivares, Adrian O; Sauer, Robert T et al. (2011) Single-molecule protein unfolding and translocation by an ATP-fueled proteolytic machine. Cell 145:257-67
Sauer, Robert T; Baker, Tania A (2011) AAA+ proteases: ATP-fueled machines of protein destruction. Annu Rev Biochem 80:587-612
Nager, Andrew R; Baker, Tania A; Sauer, Robert T (2011) Stepwise unfolding of a ? barrel protein by the AAA+ ClpXP protease. J Mol Biol 413:4-16
Davis, Joseph H; Rubin, Adam J; Sauer, Robert T (2011) Design, construction and characterization of a set of insulated bacterial promoters. Nucleic Acids Res 39:1131-41

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