The long-term objective of this research program is to understand how the sequence of a protein determines its structure, stability, folding kinetics, and ability to be denatured spontaneously and by enzymes. This is one of the central, unsolved problems in molecular biology and is important because protein folding is a prerequisite of most biological processes, and protein unfolding and misfolding often lead to disease. In addition, much of the promise of biotechnology for improving human health depends on our eventual ability to manipulate the function of proteins by altering their sequences and structures. We will probe fundamental issues in protein folding and design using P22 Arc repressor and """"""""switch"""""""" Arc, a variant in whichthe (3-sheet of the wild-type protein is replaced by a pair of helices. Arc provides an excellent model system for studying the folding and association of a single-domain dirneric protein using genetic, biochemical, and biophysical methods. Switch Arc provides an opportunity to study the sequence determinants of structural specificity and the stepwise evolution of a protein fold. These studies will be important for testing and refining our understanding of the sequence determinants of protein structure and stability, and for evaluating how well computational algorithms serve to explain and/or predict the effects of sequence changes on structure and stability. Some proteins and assemblies are too stable for spontaneous unfolding/dissociation to be a biologically relevant reaction, and energy-dependent disassembly chaperones play critical intracellular roles in dismantling such complexes, in solubilizing aggregates, and in mediating protein degradation. To begin to understand these processes, we will dissect the biochemical and enzymatic properties of ClpX?a hexameric-ring ATPase?which catalyzes protein denaturation and regulates the activity of the ClpXP protease. By studying ClpX transactions with a large number of different protein substrates, we will determine how the local stability and dynamical probities of these substrates affect enzyme-mediated denaturation. Enzymes related to ClpX are important virulence determinants in some pathogenic bacteria, play roles in regulation of bio-film formation, and function in key processes in mammalian cells, including proteasome-mediated protein degradation and vesicle fusion.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AI015706-32
Application #
7799795
Study Section
Special Emphasis Panel (NSS)
Program Officer
Park, Eun-Chung
Project Start
1979-05-01
Project End
2012-04-30
Budget Start
2010-05-01
Budget End
2011-04-30
Support Year
32
Fiscal Year
2010
Total Cost
$480,521
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
001425594
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 :
Stinson, Benjamin M; Nager, Andrew R; Glynn, Steven E et al. (2013) Nucleotide binding and conformational switching in the hexameric ring of a AAA+ machine. Cell 153:628-39
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
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
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
Davis, Joseph H; Baker, Tania A; Sauer, Robert T (2011) Small-molecule control of protein degradation using split adaptors. ACS Chem Biol 6:1205-13
Sauer, Robert T; Baker, Tania A (2011) AAA+ proteases: ATP-fueled machines of protein destruction. Annu Rev Biochem 80:587-612

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