Files in this item

FilesDescriptionFormat

application/pdf

application/pdfDAVE-DISSERTATION-2017.pdf (13MB)Restricted Access
(no description provided)PDF

Description

Title:In vitro and in vivo protein folding under stress
Author(s):Dave, Kapil
Director of Research:Gruebele, Martin
Doctoral Committee Chair(s):Gruebele, Martin
Doctoral Committee Member(s):Gennis, Robert B; Leckband, Deborah E; Kuhlman, Thomas E
Department / Program:School of Molecular & Cell Bio
Discipline:Biophysics & Computnl Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Protein folding, WW domain, Temperature jump
Abstract:Proteins are subject to a variety of stresses in biological organisms, including pressure and temperature, which are the easiest stresses to simulate by molecular dynamics simulations. The thesis will focus on discussing the effect of pressure and thermal stress on proteins including some of the fast-folding model proteins, who’s in vitro folding can be fully simulated on computers and compared directly with experiments. Pressure and temperature are prototypical perturbations that illustrate how close many proteins are to instability, a property that cells can exploit to control protein function. I will conclude with some recent in-cell experiments, and progress being made in measuring protein stability and function inside live cells under high pressure conditions. In chapters 2 and 3, fast-folding WW domains were studied (best-characterized systems for comparing experiments with simulations) by T-jump relaxation in conjunction with protein engineering. Chapter 1 is a comprehensive data set of mutational Φ-values (ΦM) as indicators for folding transition-state structure of 65 side chain, 7 backbone hydrogen bond, and 6 deletion and /or insertion mutants within loop 1 of the 34-residue hPin1 WW domain. We probed the robustness of the two hydrophobic clusters in the folding transition state, and discussed how local backbone disorder in the native-state can lead to non-classical ΦM‐values (ΦM > 1) in the rate-determining loop 1 substructure, and conclusively identify mutations and positions along the sequence that perturb the folding mechanism from loop 1-limited toward loop 2-limited folding. In chapter 2 we mutated the FBP 28 WW domain (formin-binding protein; Leu26 by Asp26 or Trp26) to alter the folding scenario from three-state folding toward two-state or downhill folding at temperatures below the melting point of the protein. The investigation was conducted using a combination of simulations over a broad temperature range with experimental temperature-jump data. Chapter 4 is focused on how attaching fluorescent protein tags to a host protein in vitro has a large non-additive effect on its folding free energy. We compared an unlabeled, three singly-labeled, and a doubly-labeled enzyme PGK (phosphoglycerate kinase). Two mechanisms for non-additivity were proposed. In the “quinary interaction” mechanism, two tags interact transiently with one another, relieving the host protein from unfavorable tag–protein interactions. In the “crowding” mechanism, adding two tags provides the minimal crowding necessary to overcome destabilizing interactions of individual tags with the host protein. Both of these mechanisms affect protein stability in cells; they must also be considered for tagged proteins used for reference in vitro. In Chapter 5 we showed that the protein unfolding/refolding reaction can be driven by a periodic thermal excitation above the reaction threshold. We were also able to speed up the reaction from an undetectable to a detectable rate by the addition of artificial thermal noise. A maximum in the recovered signal as a function of thermal noise was seen, a stochastic resonance. The study alluded that correlated noise is a physically and chemically plausible mechanism by which cells could modulate biomolecular dynamics during threshold processes such as signaling. Chapter 6 explores folding competing with misfolding or aggregation on the μs time scale using tethered WW domains. Tethered protein construct was engineered by linking two or more copies of the fast folding Fip35 WW domain with a flexible linker. We observed that adding more monomer units led to thermodynamic destabilization and slower folding rates, along with an abrupt onset of protein-protein interaction. Kinetics were determined by performing ultrafast laser temperature jump experiments at different temperatures and denaturant concentration. A simple multimeric network model is also proposed for globally fitting the thermodynamics and kinetics data. In the final chapter 7 of this thesis folding of an enzyme phosphoglycerate kinase (PGK) was studied under high pressure stress in different bacterial cytoplasm. The motivation was to understand how cell is capable of modulating the stability of its proteome when subjected to external stress especially high hydrostatic pressure. The thermodynamic stability of PGK was measured in two different strains Wildtype MG1655 and known pressure resistant J1 strain. These results were compared to in vitro experiments to reveal that cellular environment has an overall stabilizing effect on the protein thermodynamic stability but different cellular cytoplasm doesn’t affect the stability of PGK significantly.
Issue Date:2017-06-30
Type:Thesis
URI:http://hdl.handle.net/2142/98247
Rights Information:Copyright 2017 Kapil Dave
Date Available in IDEALS:2017-09-29
Date Deposited:2017-08


This item appears in the following Collection(s)

Item Statistics