Protein folding in living cells and under pressure

Protein folding;Protein folding in vivo;WW-domain;pressure;thermodynamics;kinetics;live-cell microscopy;fluorescence microscopy;Resorufin-based Arsenical Hairping Binder (ReAsH);Fluorescence (Förster) Resonance Energy Transfer (FRET);cell-cycle;pressure jump;temperature jump;p53;tetracysteine tag

Wirth, Anna J

关键词: Protein folding;    Protein folding in vivo;    WW-domain;    pressure;    thermodynamics;    kinetics;    live-cell microscopy;    fluorescence microscopy;    Resorufin-based Arsenical Hairping Binder (ReAsH);    Fluorescence (Förster) Resonance Energy Transfer (FRET);    cell-cycle;    pressure jump;    temperature jump;    p53;    tetracysteine tag;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/88149/WIRTH-DISSERTATION-2015.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Protein folding, the process through which proteins gain their functional structure, can be approached from the perspective of many disciplines. Starting with biology, we can probe how protein structure relates to function and consider how the fold of a protein interacts with the biological environment. From the chemical perspective, we can treat protein folding as a chemical reaction and study the thermodynamics and kinetics of the structural transition. With physics, we can understand the underlying forces that give rise to protein folding and use theory and simulation to describe the protein folding process on an atomic level. This thesis studies protein folding through the lens of all three of these fields with two interdisciplinary methodological themes: one at the interface of chemistry and physics and the other at the interface of biology and chemistry.In section 1, we study in detail the kinetics of fast-folding reactions following pressure-jump perturbation and pair experiment with molecular dynamics simulations. The first chapter is a review of the effects of pressure on the structure of biomolecules as well as a brief literature review of pressure-probed protein folding kinetics. We see that the methodology to study pressure-jumps is generally limited by time-scale—very fast folding is hard to study by pressure—and chapter two presents an overview of a fast pressure-jump instrument that meets this challenge. Although this instrument was developed by the previous generation of graduate students, several significant improvements are summarized in the chapter with a detailed user manual for the instrumentation. Closing up the section, we use the fast pressure-jump instrumentation as well as temperature-jump instrumentation to study the microsecond pressure and temperature-jump refolding kinetics of the engineered WW domain FiP35, a model system for beta sheet folding. With a full complement of molecular dynamics experiments mimicking experimental conditions, we show that simulation and experiment are consistent with a four-state kinetic mechanism and highlight FiP35’s position at the boundary where activated intermediates and downhill folding meet.Section 2 focuses on the interface of biology and chemistry, where we study how the protein folding reaction is impacted by immersion in the crowded intracellular environment and explore whether perturbations to the intracellular folding landscape can be linked to protein function. A review of the forces at play in the intracellular environment and the role that ultra-weak “quinary” interactions play inside living cells is presented in chapter 4, which also includes a review of the most recent literature studying biomolecular dynamics in their native environments. In chapter 5 we study the time-dependence of protein folding inside living cells as probed by live-cell fluorescent microscopy. We find that both the rate of folding and the thermodynamic stability of yeast phosphoglycerate kinase (PGK-FRET) are cell cycle-dependent, a process strictly regulated in time, suggesting that the interplay between the intracellular environment and proteins may impact their function. In chapter 6, a new probe to study protein folding in the cell is explored, namely the GFP/ReAsH Forster resonance energy transfer (FRET) pair. We show that this FRET pair suffers from bleaching artifacts but that directly excited ReAsH is an appealing prospect for studying protein folding in living cells on fast and slow time-scales. Finally, chapter 7 builds on the work presented in chapter 5 and chapter 6 by seeking a protein candidate whose function and in cell folding dynamics are linked. Several constructs of p53, a transcription factor, are explored as potential candidates for answering the question of whether protein activity level indeed can correlate with stability in living cells.