388697 Single-Molecule Conformational Motion and Atpase Activity of a AAA+ Proteolytic Machine

Wednesday, November 19, 2014: 2:24 PM
205 (Hilton Atlanta)
Harris W. Manning1, Yongdae Shin2, Benjamin M. Stinson3, Andrew R. Nager4, Tania A. Baker5, Robert T. Sauer3 and Matthew Lang6, (1)Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, (2)Massachusetts Institute of Technology, Massachusetts, MA, (3)Department of Biology, MIT, Cambridge, MA, (4)Stanford University, Stanford, CA, (5)Howard Hughes Medical Institute, (6)Chemical and Biomolecular Engineering / Molecular Physiology and Biophysics, Vanderbilt University

ClpXP is a hexameric AAA+ protease that binds, denatures and degrades protein substrates tagged for removal from the cell. Despite the many studies done on the ClpX unfoldase, it is still unknown how this motor couples ATP hydrolysis to mechanical movements to unravel a native protein and spool the polypeptide track into the ClpP degradation chamber. We used single-molecule fluorescence spectroscopy to investigate the conformational motions and ATPase activity within a single ClpX subunit. By using short-range fluorescence quenchers, we were able to resolve angstrom-level distance changes between domains and witness binding events of ATP analogs. This mechanistic study of ClpX helps to broaden our understanding of the large family of AAA+ motors and the novel techniques used here will advance the capabilities of single-molecule fluorescence measurements.

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See more of this Session: Protein Structure, Function, and Stability I
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division