All ETDs from UAB

Advisory Committee Chair

Aaron L Lucius

Advisory Committee Members

Christie Brouillette

Suzanne E Lapi

Peter E Prevelige

Charles L Turnbough Jr

Document Type


Date of Award


Degree Name by School

Doctor of Philosophy (PhD) College of Arts and Sciences


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.



To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.