Year of Award

2009

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Chemistry

Other Degree Name/Area of Focus

Biochemistry

Department or School/College

Department of Chemistry and Biochemistry

Committee Chair

J. B. Alexander Ross

Commitee Members

Bruce E. Bowler, Klara Briknarova, Michael DeGrandpre, Michele A. McGuirl

Keywords

alkaline conformational transition, electron transfer, Iso-1-cytochrome c, omega-loop D, proline isomerization

Publisher

University of Montana

Abstract

Protein folding is important because all proteins must fold to achieve their active conformer. In many cases, misfolding is the cause of disease and thus, understanding folding may lead to cures for disease. This thesis focuses on the dynamics and thermodynamics of partially unfolded states of proteins that lie near the bottom of a folding funnel. Various studies have provided evidence that partially unfolded proteins can be key intermediates in metabolic processes and in aggregation diseases. Partially unfolded proteins also provide insight into the types of structures that guide/misguide the process of protein folding. Less is known about how stability affects the roughness of an energy landscape at the bottom of the folding funnel. Histidine-heme alkaline conformers of cytochrome c are used as a model for a late folding intermediate or partially unfolded state that can be exploited to study roughness near the bottom of a folding funnel.

In my thesis work, I have developed a novel method using electron transfer (ET) reactions to probe these conformational changes and thus provide insight into the dynamics of late folding processes not available through standard stopped-flow methods. This method has helped to probe two factors that affect dynamics near bottom of funnel: overall stability and the position of a residue in a loop that stabilizes a misfold. The main findings from my work include: a decrease in stability decreases the roughness of a folding funnel and changing the position of a residue responsible for misfolding strongly affects stability and dynamics of the misfolded state. The robustness pertaining to the gated ET method highlighted in this work is that it allows extraction of discrete rate constants for conformational changes under conditions where these rate constants cannot usually be measured directly. The method has also been applied to the dynamics of proline isomerization. The data demonstrate that rates of ET in proteins can be tuned efficiently using a combined strategy of modulating the sequence position and nature of the metal ligand involved in conformational gating. Thus, ET gates can be readily tuned for metabolic processes or the development of molecular switches.

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© Copyright 2009 Swati Bandi