Advisory Committee Chair
Aaron L Lucius
Advisory Committee Members
Christie Brouillette
Suzanne E Lapi
Peter E Prevelige
Charles L Turnbough Jr
Document Type
Dissertation
Date of Award
2018
Degree Name by School
Doctor of Philosophy (PhD) College of Arts and Sciences
Abstract
E. coli ClpA is an AAA+ (ATPase Associated with diverse cellular Activities) chaperone that catalyzes the ATP-dependent unfolding and translocation of substrate proteins targeted for degradation into a protease, ClpP. ClpA, like many other AAA+ proteins, assembles into a hexameric ring competent for binding polypeptide substrate clients in the presence of ATP. Each ClpA protomer contains two nucleotide binding domains, NBD1 and NBD2. Recently, our lab applied hydrodynamic techniques to quantify the self-assembly mechanism of a closely related AAA+ chaperone, E. coli ClpB. Here we apply the techniques and analysis from that work to investigate the nucleotide-linked self-assembly mechanism of ClpA. In this work, sedimentation velocity studies are used to quantitatively examine the ClpA self-assembly mechanism in the absence of nucleotide for wild type ClpA (ClpAWT) and various ClpA variants shown to be deficient at hydrolyzing ATP at one or both NBDs. We observe differences in the resulting self-assembly equilibrium constants obtained for ClpAWT relative to that obtained for each ClpA variant. In that work we showed that changes to the primary sequence of the proteins could perturb the assembly mechanism. Sedimentation velocity studies were also performed on ClpA in the presence of ATP using variants deficient in ATP hydrolysis at both NBDs. The stoichiometry and affinity of nucleotide binding to NBD1 and NBD2 are revealed by examining the dependence of the apparent association equilibrium constants on nucleotide concentration. In this work we show that in the presence of ATP, ClpA resides in a distribution of monomers, dimers, tetramers, hexamers, and dodecamers. Average stoichiometries and affinity for ATP binding to each oligomer are revealed from modeling the apparent equilibrium constant for dimerization, tetramerization, hexamerization, and dodecamerization of ClpA as a function of free nucleotide concentration.
Recommended Citation
Duran, Elizabeth C., "Examination Of The Dynamic Assembly Mechanism Of E. Coli Clpa" (2018). All ETDs from UAB. 1565.
https://digitalcommons.library.uab.edu/etd-collection/1565
