Advisory Committee Chair
Aaron L Lucius
Advisory Committee Members
James C Patterson
Peter E Prevelige
David A Schneider
Jun Zhang
Document Type
Dissertation
Date of Award
2020
Degree Name by School
Doctor of Philosophy (PhD) College of Arts and Sciences
Abstract
ClpA, a Class I AAA+ (ATPases Associated with various cellular Activities) motor, as-sembles into a tiered hexamer containing two AAA+ nucleotide binding domains (NDBs), D1 and D2. These NBDs couple nucleotide triphosphate binding and hydrolysis to mechanical work that drives polypeptide unfolding and translocation. Here we study the kinetic mechanisms of polypeptide translocation at the two domains to elucidate their contributions to the overall wildtype activity of ClpA. We subjected Walker B variants of ClpA that lack ATPase activity in one of the two domains to single-turnover stopped-flow techniques using a new fluorescence anisotropy and total fluorescence method. We found that the two domains translocate poly-peptide substrate with unique rates and similar kinetic step-sizes at saturating ATP. D2 was found to support the majority of ClpA’s translocation activity, with D1 constituting less than 1% of WT activity. Both D1 and D2 were found to traverse approximately 15 aa per repeating cycle of translocation, similar to WT. Moreover, we showed that both variants exhibited positive cooperativity with respect to ATP binding indicating the presence of inter-monomer interac-tions within hexameric ClpA.
Recommended Citation
Scull, Nathaniel, "Comparative Analysis Of The Translocation Kinetics For Clpa’S Atpase Domains 1 & 2" (2020). All ETDs from UAB. 2935.
https://digitalcommons.library.uab.edu/etd-collection/2935
