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Title:Cellular influence on protein folding
Author(s):Gelman, Hannah Madeline
Director of Research:Gruebele, Martin
Doctoral Committee Chair(s):Kuhlman, Thomas E.
Doctoral Committee Member(s):Freeman, Brian; Luthey-Schulten, Zaida A.
Department / Program:Physics
Discipline:Physics
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):fluorescence microscopy
protein folding
biophysics
chaperones
Fӧrster Resonance Energy Transfer (FRET)
Abstract:Advances in the study of protein folding and structure have greatly expanded our understanding and ability to predict and modify protein structure and, increasingly, even function. Attention has now turned to developing a better understanding of how protein structure, function, and folding interact to enable necessary biochemical processes in the cell. This work includes efforts to better understand the folding of model proteins in vitro, with an eye towards how this insight can inform questions about protein dynamics in more complex environments and protein-protein interactions. In addition, recent advances in NMR, mass spectrometry, fluorescence microscopy, and other techniques have enabled the study of protein folding in the cellular environment and have shown that the effect of the cell on protein folding is variable and difficult to predict. Researchers continue to develop new tools to investigate the effect of the cellular environment on more complicated biomolecular systems. This thesis is roughly divided into two sections: Chapters 1-2 discuss fast protein folding in vitro and Chapters 3-6 address the study of protein folding and folding mediated processes in the cell. Chapter 1 is a survey of the theory of protein folding and the major techniques and findings from the study of fast folding proteins in vitro, with a special emphasis on how this work informs our understanding of more complex protein dynamics. Chapter 2 characterizes dodine, a compound that combines the properties of chatropic denaturants and detergents. Chapter 3 is an introduction to how perturbative methods developed in vitro can be applied to study processes in the cell that are inaccessible by conventional steady-state measurements. Chapter 4 is a practical guide to improving the accuracy and reliability of Fluorescence Relaxation Imaging (FReI), our Fӧrster Resonance Energy Transfer (FRET) microscopy based technique for studying biomolecular kinetics in the cell. Chapter 5 describes the development and characterization of the fluorescent construct GPGK-tc and its use to study population-level variation of protein folding in E. coli. Finally, Chapter 6 examines the interaction of the chaperone Hsp70 with an unfolding substrate using a FRET-based binding assay both in vitro and in cells.
Issue Date:2015-07-14
Type:Thesis
URI:http://hdl.handle.net/2142/88268
Rights Information:Copyright 2015 by Hannah Gelman
Date Available in IDEALS:2015-09-29
Date Deposited:August 201


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