Characterization of ClpA-substrate interaction time by single molecule fluorescence microscopy

BIOL 24

Mary E Farbman, farbman@mit.edu1, Anne Gershenson, gershenson@brandeis.edu2, and Stuart Licht, lichts@mit.edu1. (1) Department of Chemistry, Massachusetts Institute of Technology, 32 Vassar Street, Building 56-522, Cambridge, MA 02139, (2) Department of Chemistry, Brandeis University, MS 015, 415 South St, Waltham, MA 02454
The bacterial ClpAP protein complex is a macromolecular machine responsible for proteolytic degradation of certain tagged protein substrates. ClpA unfolds and processively translocates its substrates into the ClpP proteolytic pore in an ATP-dependent manner. The eleven amino acid “ssrA” AANDENYALAA sequence at the carboxy terminus is sufficient to target substrate proteins to the ClpAP complex. We have used single molecule fluorescence microscopy to further study the interactions between immobilized ClpA oligomers and the ssrA peptide. Epitope-tagged ClpA molecules were affixed to an antibody-derivatized glass surface. The interactions of a Cy3-labeled ssrA peptide with the surface-associated ClpA were observed in the presence of ATP or a non-hydrolyzable ATP analog using total internal reflection microscopy. From the observed distribution of residence times and apparent association rates, we report estimates for the microscopic rate constants of substrate processing by ClpA.
 

Frontiers in Chemical Biology
5:00 PM-7:00 PM, Sunday, August 19, 2007 BCEC -- Exhibit Hall - B2, Poster

Division of Biological Chemistry

The 234th ACS National Meeting, Boston, MA, August 19-23, 2007