Date of Award
2023-12-01
Degree Name
Doctor of Philosophy
Department
Chemistry
Advisor(s)
Ricardo A. Bernal
Abstract
Chaperonins are large protein complexes responsible for the refolding of misfoldedsubstrate. Many mechanisms of the folding process have not been completely elucidated. Here we perform biochemical and structural analysis on two chaperonins, the human mitochondrial Hsp60 and the bacteriophage EL encoded PhiEL, to better understand key structural components and their purpose to the chaperonin function. Hsp60 plays an essential role in maintaining protein within the mitochondrial matrix. Despite its importance, the detailed mechanism of ATP hydrolysis and the role of the C-terminal tail remains unresolved. Here we show that the C-terminal tail not only acts as a sensor for the arrival of substrate into the refolding chamber but also protects against oxidative damage from reactive oxygen species. Our results reveal that removal of the C-terminal tail leads to a stalled chaperonin unable to sense and refold misfolded substrate. High resolution reconstructions reveal the detailed mechanism of ATP hydrolysis where access to the γ-phosphate is blocked by the carboxylate oxygen of an aspartate residue. Binding of the C-terminal tail to substrate displaces this oxygen and allows ATP hydrolysis. These results bring a better understanding of how Hsp60 functions at an atomic level where substrate arrival activates ATP hydrolysis through an allosteric trigger. Ring separation is a unique aspect utilized by few chaperonins in the refolding of denatured substrate. Previously seen natively only in Hsp60, this ability has recently been identified in the phage EL encoded PhiEL chaperonin. In this study we examine the effects of introducing a T91A point mutation to PhiEL to inhibit ring separation. Our results reveal that the locking of the double ring complex in PhiEL reduces its chaperonin function by limiting the substrates that can be refolded. Weâ??ve obtained cryo-EM data of the T91A mutant as well as the wild-type PhiEL to understand how the locking of the double ring affects this change in the folding function. These results bring a better understanding of how ring separation plays such a key role in the protein folding mechanism of chaperonins.
Language
en
Provenance
Recieved from ProQuest
Copyright Date
2023-12
File Size
95 p.
File Format
application/pdf
Rights Holder
Daniel Han von Salzen
Recommended Citation
von Salzen, Daniel Han, "Structural Studies Of The Human Hsp60 And Bacteriophage Phiel Chaperonins" (2023). Open Access Theses & Dissertations. 4031.
https://scholarworks.utep.edu/open_etd/4031