All ETDs from UAB

Advisory Committee Chair

Aaron L Lucius

Advisory Committee Members

Margaret A Johnson

Eugenia Kharlampieva

Kirill M Popov

David A Schneider

Document Type


Date of Award


Degree Name by School

Doctor of Philosophy (PhD) College of Arts and Sciences


E. coli ClpB and S. cerevisiae Hsp104 help cells survive and adapt to environmental stress. Extreme stress denatures proteins, causing toxic aggregates to form. ClpB/Hsp104 resolve aggregates, returning damaged proteins to their proper fold and function or releas-ing them for degradation. This function, broadly defined as protein remodeling, is com-mon to AAA+ proteins. This superfamily also shares common structural elements includ-ing Walker A and Walker B motifs in the nucleotide binding domain(s). ClpB/Hsp104 have two NBDs, similar to ClpA. ClpB/Hsp104 also have a middle (M) domain, unique to the disaggregases. Because disaggregases do not covalently modify their substrates, monitoring the progress their reactions has been challenging. Without a method of moni-toring translocation in the absence of covalent modification, ClpB/Hsp104 have been as-sumed to operate via the same molecular mechanism as ClpA. That mechanism is proces-sive translocation or complete threading. Our lab developed a single-turnover transient state kinetic assay to study ClpA in the presence or absence of its proteolytic partner. Re-cently, our lab applied this strategy to study the molecular mechanism of ClpB. In a para-digm shifting finding, we determined that ClpB is a non-processive translocase, taking just one or two kinetic steps on a polypeptide substrate. Here, we expand this work in two directions. First, we extend this investigation to the eukaryotic homologue, Hsp104. Our study of Hsp104 required foundational investigations of its assembly state and pep-tide binding capabilities. With that information, we were able to apply our single-turnover transient state kinetic assay. We found that Hsp104 is also a non-processive translocase, partially threading polypeptide substrate through the Hsp104 axial channel. We also es-tablished that a variant with increased disaggregation and neuroprotection phenotypes was slower to dissociate from substrate than the wild type protein. Second, we built on our recent insights into the ClpB molecular mechanism by investigating how the co-chaperone DnaK affects the ClpB mechanism. Surprisingly, we discovered that DnaK acts as a peptide release factor, causing ClpB to dissociate from polypeptide substrate. With these findings, we are now poised to further extend the investigation of the Hsp104 molecular mechanism to include the effect of the co-chaperone Hsp70.



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