All ETDs from UAB

Advisory Committee Chair

Aaron L Lucius

Advisory Committee Members

David E Graves

Donald D Muccio

Peter E Prevelige

Bingdong Sha

Document Type

Dissertation

Date of Award

2015

Degree Name by School

Doctor of Philosophy (PhD) College of Arts and Sciences

Abstract

As a member of the Clp/Hsp 100 chaperone family, Escherichia coli ClpB is able to disaggregate denatured proteins with assistance of DnaKJE co-chaperones to help cell survive under stress. However, the working mechanism of ClpB disaggregation activity remains unclear. The active structure of ClpB is shown to be a hexameric ring and ATP binding and hydrolysis are required for ClpB to perform its chaperone activity. Thus, studying the energetics and kinetics of the ATP linked ClpB assembly equilibrium is essential for the quantitative examination of ClpB - protein substrate interaction to fully reveal its disaggregation mechanism. ATPgS (a slowly hydrolysable ATP analog) is used as a model for ATP in this study. In order to examine the ligand - linked ClpB assembly, ClpB self-assembly in the absence of nucleotide needs to be determined. In the first part of this study, we introduce to you the methods that were applied to perform this study using analytical ultracentrifugation. By performing NLLS analysis of the simulated sedimentation velocity data, we presented that both thermodynamic and kinetic parameters of a complex assembly system can be determined accurately with certain limitations. Further, the linkage of ligand binding can be determined by analyzing the assembly equilibrium constants as a function of [ligand]. In the second part, we applied the methods discussed in the first section into the determination of the assembly energetics and kinetics for ClpB in the absence of nucleotide. Here, we show that ClpB can form hexamers in the absence of nucleotide through two intermediates, dimers and tetramers. The assembly equilibrium constants and dissociation rate constants were determined for each oligomer in the absence of nucleotide. With the result of that, we examined the linkage of [ATPgS] binding to ClpB assembly. Not like assumed in many studies that ClpB forms hexamer only in the presence of ATP/ATPgS, here we show that ClpB exhibits a dynamic equilibrium in the presence of both limiting and excess ATPgS. ClpB monomer, dimer, tetramer, and hexamer were observed and their assembly equilibrium constants were determined. These interaction constants make it possible to predict the concentration of hexamers present and able to bind to co-chaperones and polypeptide substrates. Such information is essential for the interpretation of many in vitro studies. Moreover, the ATPgS bind equilibrium constant and stoichiometry for each oligomer were determined for the first time. All twelve NBDs of the hexameric ring are saturated with ATPgS binding, however, the binding stoichiometry of dimers and tetramers is one fewer than the maximum number of the NBDs, which suggests an open conformation. Our results are constant with the previously published structure studies. Finally, the strategies presented here are broadly applicable to a large number of AAA+ molecular motors that assemble upon nucleotide binding.

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