Date of Award

12-2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Advisor(s)

Mark S. Braiman

Keywords

Anions, Citrate, Intracellular loop 1, Precipitation, Proteorhodopsin, Purification

Subject Categories

Chemistry

Abstract

Proteorhodopsin (pR) is an intrinsic membrane protein with an important role in solar-energy storage of the biosphere. Earlier work in our lab has shown that polyhistidine-tagged pR can be purified by means of selective precipitation with citrate under specific conditions, as can a number of mutants based on this His6-tagged pR. Purification of a heterologously-expressed trans-membrane protein by a simple salt such as citrate is novel. However, such a phenomenon leads to several questions: How does citrate cause pR precipitation? Does the polyhistidine-tag assist in such a precipitation? Is this precipitation pH-specific? Does citrate affect the function of pR? Does citrate-induced pR precipitation have any biological significance? Are there other ions that could cause pR precipitation?

This dissertation focuses on understanding the nature of the interaction of pR with citrate and other anions, and in particular on trying to take advantage of this interaction in order to develop a novel membrane protein purification method. The end goal that branches out of these two aims is to utilize the compact citrate interaction site identified in pR, by incorporating it into other membrane proteins and using it to permit their purification by similar simple procedures.

In Chapter 1, I briefly provide some background information on the wide variety of structurally-similar proteins as rhodopsins that include pR. I also describe the general importance of developing purification methods for 7-helix membrane proteins, including pR.

Chapter 2 focuses on the investigation of the nature of citrate-binding site of pR. To address the main question of how citrate aids in pR purification, site-directed mutagenesis technique was applied to generate several single, double, triple or quadruple mutants of pR in a histidine-tag free background, which were then tested for their reactivity to citrate. Several different anions were tested to examine if precipitation of pR was specific to citrate or whether the precipitation is susceptible to other negatively charged salts. Photocycle of pR progresses through several intermediates, each with a distinct absorption maximum (described in subsection 1.3.1). M-intermediate is detected at pH ≥ 8 with λmax = 410 nm. Flash spectroscopy involves excitation of pR at a particular wavelength that leads to transient absorption, thus, signaling the formation of the corresponding intermediate. Flash-induced transient visible absorption measurements were used to assay the effect of exposing pR to citrate on its physiological function.

Chapter 3 describes the development of a method of purification of pR using simple salts, citrate and phosphate. Chapter 4 begins an exploration of a future direction. The ultimate objective is to apply the above techniques for the general purpose of 7-helix membrane protein purification, especially for the important class of pharmacological receptors known as GPCRs. An attempt at heterologous expression in E. coli, and purification, of a mammalian GPCR, is described therein. Such a method would be desirable for obtaining proteins for structural, functional and pharmacological studies.

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