All ETDs from UAB

Advisory Committee Chair

Aaron L Lucius

Advisory Committee Members

David E Graves

Kirill M Popov

Christie G Brouillette

Matthew B Renfrow

Document Type


Date of Award


Degree Name by School

Doctor of Philosophy (PhD) College of Arts and Sciences


ATP-dependent proteases catalyze the removal of both misfolded and properly folded proteins in cellular quality control pathways. ClpAP shares a structural homology with other ATP-dependent proteases where a hexameric ring of ClpA associates with one or both ends of the cylindrically-shaped protease ClpP, which contains serine protease active sites sequestered in its inner core. ClpA contains two nucleotide binding domains where ATP is bound and hydrolyzed, termed Domain 1 (D1) or 2 (D2). D1 has been shown to be primarily responsible for ClpA oligomerization, but both domains appear to support polypeptide translocation independently. We previously reported a molecular mechanism for ClpA catalyzed polypeptide translocation in the absence of ClpP, including estimates of the elementary rate constant, the overall translocation rate, and the kinetic step-size. We have applied here a single-turnover stopped-flow fluorescence method to examine polypeptide translocation catalyzed by ClpA in both the presence and absence of the proteolytic component ClpP. We propose models for ClpA and ClpAP catalyzed pol-ypeptide translocation where D1 or D2 limits the rate of ClpA catalyzed polypeptide translocation when ClpP is either absent or present, respectively. In our models, the ob-served rate-limiting step occurs immediately after an ATP binding event in a cycle of translocation. For ClpA, this step repeats every ~14 amino acids translocated with an ob-served rate constant of ~1.39 s-1, whereas for ClpAP this step repeats every ~2 - 5 amino acids translocated with an observed rate constant of ~6.6 s-1. However, our model for ClpAP catalyzed polypeptide translocation was based on data collected for conditions where a mixture of ClpAP complexes was favored. Thus, it was unclear whether ClpAP complexes with one or two associated ClpA hexamers translocate polypeptide with the same mechanisms. To address this, we report here an examination of the dependence of the mechanism for ClpAP catalyzed polypeptide translocation on the ClpAP species dis-tribution. We conclude that ClpAP complexes with one or two ClpA hexamers associated translocate polypeptide with identical mechanisms. Therefore, our data are consistent with a model where the rate-limiting step for translocation is coupled to ATP hydrolysis at D2 for all ClpAP complexes.