All ETDs from UAB

Advisory Committee Chair

Aaron L Lucius

Advisory Committee Members

David E Graves

N Patrick Higgins

Donald D Muccio

Bingdong Sha

Document Type

Dissertation

Date of Award

2013

Degree Name by School

Doctor of Philosophy (PhD) College of Arts and Sciences

Abstract

Both E. coli ClpA and E. coli ClpB belong to the Clp/Hsp100 chaperone family. They share many common features, such as the same conserved motifs (Walker A, Walker B, Arginine finger, Pore loop, etc.), hexameric ring-shaped structure, and double nucleotide binding domains. ClpA and ClpB are closely related to each other. However, they participate in different protein quality control pathways. ClpA regulates cellular functions by either associating with ClpP to form the ClpAP complex (an ATP-dependant protease) to degrade unwanted polypeptides or remodeling inactive proteins to their biological active forms. ClpA can recognize specific sequences located on either N- or C-terminus of its substrates and unstructured polypeptides. On the other hand, ClpB cannot associate with any known peptidase for protein degradation, but it can catalyze protein disaggregation with or without the assistance of DnaK chaperone system. First part of my study focuses on quantitatively investigating the binding specificity, affinity and stoichiometry for both ClpA and ClpB binding to tagged or untagged substrates using steady-state anisotropy titrations. Our results suggest that ClpA can specifically recognized SsrA tag, but ClpB cannot. Both ClpA and ClpB nonspecifically bind to unstructured polypeptides. Multiple ClpA or ClpB hexamers are involved in binding to long unstructured polypeptide substrates. And they both require the substrate with a minimum length for an optimal binding. The second part of my study mainly focuses on understanding the mechanism of ClpB catalyzed protein disaggregation. It is well known that ClpA is able to processively translocate polypeptide through the central channel of its hexamer. Partly, based on their structural similarities, the hypothesis that ClpB translocates substrate through the axial channel to catalyze protein disaggregation has been proposed for many years. However, data from our single-turnover fluorescence stopped-flow experiments and single-turnover FRET stopped-flow experiments suggest that, unlike ClpA, ClpB is NOT a processive translocase. Our studies reveal different stories on substrate interaction for ClpA and ClpB. With our results in mind, we propose the ClpB catalyzes protein disaggregation by binding to protein aggregates, exerting force and dissociating and rebinding frequently.

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