Advisor(s)
Aaron L Lucius
Committee Member(s)
Alexa Mattheyses
David Schneider
Elizabeth Duran
Hui-Ting Lee
Tracy Hamilton
School
College of Arts and Sciences
Document Type
Dissertation
Department (new version)
Chemistry
Date of Award
1-6-2025
Abstract
Escherichia coli ClpB and Saccharomyces cerevisiae Hsp104 are part of the Hsp100 family of proteins responsible for maintaining protein homeostasis in a cell during heat-stress conditions. Structurally, ClpB and Hsp104, in their active state, assemble into hexameric ring complexes with a hollow axial channel. Functionally, these enzymes are hypothesized to couple energy from ATP binding and hydrolysis to unfold protein substrates and translocate them through their axial channel. However, the molecular mechanisms of protein unfolding and translocation catalyzed by both ClpB and Hsp104 are not well understood. In this dissertation, we report the development of a single-turnover transient state kinetics strategy on a sequential mix stopped-flow apparatus that investigates processive protein unfolding and translocation catalyzed by both ClpB and Hsp104. Our results indicate that during ClpB catalyzed protein unfolding at saturating [ATP], slow unfolding is followed by fast translocation, which indicates unfolding and translocation are two distinct kinetic processes. However, at low [ATP], translocation becomes partially rate-limiting. We report kinetic parameters such as rate, elementary rate constants and step sizes as a function of [ATP] for ClpB catalyzed protein unfolding reaction. These parameters led us to quantify the processivity of ClpB, which is independent of [ATP]. Further, we apply the same method to study the role of allosteric communication between two ATP binding sites of Hsp104 during enzyme catalyzed protein unfolding reaction. In totality, this dissertation puts forward a new fluorescence-based methodology with unique analysis tools to determine the elementary steps necessary for enzyme catalyzed protein unfolding and translocation for two structurally similar enzymes. This approach opens doors to a vast family of Hsp100 proteins to investigate their molecular mechanisms of protein unfolding and translocation.
ProQuest ID
Recommended Citation
Banwait, Jaskamaljot Kaur, "Unraveling The Kinetic Mechanisms Of Protein Unfolding And Translocation Catalyzed By Hsp100 Unfoldases" (2025). All ETDs from UAB. 7318.
https://digitalcommons.library.uab.edu/etd-collection/7318
