Advisory Committee Chair
Aaron L Lucius
Advisory Committee Members
Herbert C Cheung
David E Graves
Donald M Muccio
Peter E Prevelige
Document Type
Dissertation
Date of Award
2010
Degree Name by School
Doctor of Philosophy (PhD) College of Arts and Sciences
Abstract
Escherichia coli ClpAP, akin to other energy dependent proteases, is constructed from two distinct enzymes, a protein unfoldase, ClpA, and a protease, ClpP. ClpA is responsible for enzyme catalyzed protein unfolding and polypeptide translocation, while ClpP accepts and proteolytically degrades the newly unfolded protein. ClpA is defined as a molecular chaperone since it has been shown to remodel protein complexes in the absence of ClpP. In order for ClpA to bind protein substrates targeted for removal or remodeling, ClpA requires nucleotide binding to assemble into an oligomeric form with protein binding activity. In addition to this nucleotide driven assembly activity, ClpA self associates in the absence of nucleoside triphosphate binding. An examination of the energetics of the nucleotide driven assembly process cannot be performed without a thermodynamic model of the self-assembly process in the absence of nucleotide cofactor. Here we report an examination of the self and ligand-linked assembly properties of the Escherichia coli ClpA protein unfoldase through the application of analytical ultracentrifugation and light scattering techniques, including sedimentation velocity, sedimentation equilibrium, and dynamic light scattering approaches. An investigation of the effect of temperature on the self-association of Escherichia coli ClpA between 3.9 °C to 38.2 °C was performed using hydrodynamic techniques. This data led us to propose a monomer-dimer-tetramer equilibrium to describe the self-assembly of ClpA. Additionally, we report the assembly properties of ClpA in the presence of nucleoside di- and triphosphates and correlate the assembly state of ClpA in the presence of selected nucleotides with both polypeptide binding activity and enzymatic activity. Herein we show that all of the selected nucleoside di- and triphosphates promote the formation of hexameric ClpA. However, not all promote a form of hexameric ClpA that is active in polypeptide binding and translocation. These results suggest that characteristics of the gamma phosphate of a nucleotide may serve to switch ClpA into a conformational state with high peptide binding activity.
Recommended Citation
Veronese, Phillip Keith, "Thermodynamic and Hydrodynamic Properties of Escherichia coli ClpA Assembly and Correlation with Polypeptide Binding" (2010). All ETDs from UAB. 3217.
https://digitalcommons.library.uab.edu/etd-collection/3217
