Date

June 2007

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Biochemistry and Molecular Biology

Institution

Oregon Health & Science University

Abstract

A protein’s structure and oligomerization state dictates its function. Unfortunately, for many proteins, especially membrane-bound proteins like G-protein coupled receptors (GPCRs), elucidating the dynamic conformational changes required for activation is often not possible using traditional structural methodologies. As a result, new methodologies are needed for the study of these receptors. This dissertation discusses three things. First, it presents the development of a novel site-directed fluorescence labeling (SDFL) methodology that can be used to determine protein secondary structure at the level of the backbone fold and provide protein tertiary structural constraints of ~ 5 – 15 Å. This method, which exploits the distance-dependent quenching of bimane fluorescence by proximal tryptophan residues, is generally useful for studying protein/protein interactions as well as measuring real-time conformational changes in membrane proteins. Second, a way to automate and increase the speed with which SDFL methods can be undertaken is presented. The thiol-cleavable fluorophore, PDT-Bimane, shows solvent-sensitive characteristics to its fluorescence, as well as susceptibility to quenching by proximal tryptophan residues. Together, these properties enable its use for studying both protein secondary and tertiary structure. Furthermore, the reducible nature of PDTBimane resolves problems often faced in SDFL experiments: ensuring specific labeling of cysteine residues, determining the extent of free label contamination, and accurately determining labeling efficiency even at low sample concentrations. Thus, the ability to cleave PDT-Bimane off the protein enables automated, rapid determination of these parameters, and positions it as an ideal fluorophore for high-throughput SDFL structural studies. Finally, a spectroscopic approach is described for studying the oligomerization state of visual rhodopsin, the model GPCR. This approach uses a novel combination of SDFL and resonance energy transfer methodologies to determine that visual rhodopsin, when reconstituted into a membrane environment, prefers to self-associate into higher order oligomers. In fact, greater than 90% of the receptors were found to associate both in the dark and following light-activation. In summary, this dissertation both develops novel SDFL methods to assess protein/protein interactions and conformational changes, as well as demonstrates how to assess oligomerization states in difficult to study membrane proteins.

Identifier

doi:10.6083/M41V5BZB

School

School of Medicine

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