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Title: Experimental Investigation of Protein Stability and Protein Folding Utilizing the Glassy State
Authors: Cho, Theresa Yuek-Chiu
Advisors: Debenedetti, Pablo G
Contributors: Chemical and Biological Engineering Department
Subjects: Chemical engineering
Issue Date: 2012
Publisher: Princeton, NJ : Princeton University
Abstract: Proteins are one of the main components of living systems and have a wide diversity of structure and function. Although proteins are very useful in many different applications, the number of applications is limited by the fact that the folded and functional state of a protein is only marginally stable. Increasing the stability of proteins in general, and enzymes in particular, is of major technological importance. The first part of this dissertation research is directed at studying phenomena and factors that impact protein stability. Experiments using differential scanning calorimetry, infrared spectroscopy, ultraviolet spectroscopy and enzymatic assays were conducted to study the effect of trehalose on lysozyme aggregation when subject to thermal treatment. A high concentration of lysozyme (200 mg/ml) in a high concentration of trehalose (80 wt%) results in aggregation. On the other hand, the use of lower trehalose concentrations (0 to 40 wt%) prevents or inhibits aggregation at both high and low protein concentrations. A higher initial protein concentration increases the rate of aggregation. The rates of aggregation and inactivation of lysozyme in trehalose follow first-order kinetics. Despite the attractive features of stabilized functional proteins for biotechnological applications, proteins in vivo can misfold resulting in loss of functionality or becoming toxic to the cell. Correct folding of proteins is one of nature's most ubiquitous phenomena. Understanding the mechanisms underlying protein folding is important for providing insight into protein misfolding and aggregation as well as for the rational modification and design of novel proteins. One important step in unraveling the protein folding mechanisms is to characterize intermediate or partially structured states, in terms of their structural, kinetic and thermodynamic properties. However, uncovering the mechanisms underlying the folding process has always been a challenge due to the very short time scales for proteins to fold. The second part of this dissertation focuses on the development of a new experimental technique to study protein folding mechanisms by capturing intermediate structures during protein refolding using a quench-and-refold approach with infrared spectroscopy. The experimental procedure developed involves first thermally denaturing a protein, then trapping the unfolded biomolecule by rapidly quenching the system into a glassy state, and finally heating the system slowly across its glass transition. The main advantage of this technique is the opportunity to observe subtle structural changes as they evolve from a denatured state back to the native state over a continuum in time, due to the slow kinetics found in the viscous liquid state near the glass transition temperature. Infrared spectroscopy is employed to observe secondary structural changes throughout the procedure. This method requires careful consideration of an appropriate protein solution medium or solvent, such that the solvent does not denature the protein nor permit aggregation. The solvent should also prevent ice crystallization upon cooling or heating. A solution consisting of sucrose, ethylammonium nitrate, and water (or heavy water) was eventually chosen and used for subsequent quench and refold experiments. Lysozyme was used as the model protein for initial experiments. Further studies were then conducted with ribonuclease A. By comparison of differential scanning calorimtery and infrared spectroscopy measurements, early energy-neutral structure formation events are revealed.
Alternate format: The Mudd Manuscript Library retains one bound copy of each dissertation. Search for these copies in the library's main catalog
Type of Material: Academic dissertations (Ph.D.)
Language: en
Appears in Collections:Chemical and Biological Engineering

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