Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biochemistry & Molecular Biophysics

First Advisor

James Shorter


Hsp104 is a protein remodeling factor from Saccharomyces cerevisiae which couples the energy released from ATP hydrolysis to the disaggregation of amyloid and amorphous protein aggregates. Hsp104 assembles into a hexamer with a central channel through which aggregated substrates are threaded. The exact mechanistic details of how Hsp104 accomplishes disaggregation have not been fully articulated. In this work, our goal was to determine the role of inter-subunit communication in amyloid and amorphous substrate disaggregation and to resolve the function of the distal loop of the Middle Domain of Hsp104.

Using a mutant subunit doping analysis, we determined whether Hsp104 subunits work in concert to accomplish substrate disaggregation. We found that during amorphous substrate disaggregation, Hsp104 subunits act independent from each other. In contrast, when Hsp104 encounters more stable substrates, like amyloid, the subunits collaborate to hydrolyze ATP and to bind substrate to accomplish disaggregation. The amount of inter-subunit collaboration was found to be commensurate with the stability of the aggregated substrate: more stable aggregates required more subunit collaboration. Furthermore, we found two Hsp104 mutants, L462R and D704N, which specifically impaired inter-subunit communication, indicating that the regions containing these mutations may be critical for relaying intra-subunit signals.

To disaggregate amorphous aggregates, Hsp104 must work in concert with the chaperone pair Hsp70 and Hsp40. How Hsp104 interfaces productively with Hsp70 and Hsp40 is undetermined. Recently, it has become apparent that the coiled coil Middle Domain (MD) of Hsp104 and ClpB play a key role in collaboration with Hsp70 and Hdp40. Here, we determine that the distal loop portion of the MD is especially important for collaboration with Hsp70 and Hsp40, as mutations in this region specifically ablate the functional interaction with Hsp70. Furthermore, the distal loop of Hsp104 appears to be involved in an interface with the second nucleotide binding domain (NBD2) of Hsp104 as shown by tryptophan fluorescence, hydroxyl radical foot-printing, and disulfide cross-linking. We hypothesize that the distal loop interaction with NBD2 is inhibitory to productive collaboration with Hsp70 and Hsp40.

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