Date

1-1-2018

Document Type

Dissertation

Degree

Doctor of Philosophy

Department

Chemistry

First Adviser

Glover, Kerney J.

Abstract

The main topic of this doctoral dissertation is the biophysical characterization of caveolin-1. Caveolin-1 is an integral membrane protein that has been shown to be essential for the formation of caveolae. Caveolae are 50-100 nm invaginations in the plasma membrane that have a plethora of cellular functions including signal transduction, relieving mechano-stresses on the cell, and endocytosis. Caveolin-1 is at the center of all of the functions of caveolae and has been shown to play a predominant role in disease states. However, while there are a large number of biological studies on caveolin-1, there are few biophysical studies, leading to a lack of understanding of the structure, topology and oligomerization of caveolin-1. The progress made in these three main areas of caveolin-1 research as well as introducing a novel in vitro functional assay for caveolin-1 and a broadly applicable membrane protein isolation technique are introduced. In chapter 1, background and general information about caveolin-1 and the biophysical techniques that were utilized for its characterization are discussed. Chapter 2 discusses the structural characterization of a caveolin-1 construct containing residues 62-136 using NMR spectroscopy revealing that the N-terminal residues (62-85) were dynamic and caveolin-1 contains a helix-break-helix motif with two approximately equal length helices. Chapter 3 discusses the structural characterization of caveolin-1 residues (62-178) using NMR spectroscopy. Caveolin-1(62-178) is the longest construct of caveolin-1 to be structurally characterized and encompasses the previously uncharacterized C-terminal domain which formed a long helix. Additionally, caveolin-1 contains a helix-break-helix-break-helix motif. In chapter 4, alanine and phenylalanine scanning mutagenesis of caveolin-1 82-136, was utilized to identify key structural residues within both helix-1 and helix-2. In chapter 5, the efforts to establish an in vitro functional assay for caveolin-1 utilizing the inhibition of endothelial nitric oxide synthase is presented. In chapter 6, cysteine scanning mutagenesis was utilized to evaluate the exposure of single residues in the caveolin-1 scaffolding domain to determine the topology of caveolin-1. Additionally, an evaluation of several different maleimide probes is presented. In chapter 7, a novel method to measure membrane protein oligomerization utilizing homo-FRET in liposomes is presented. Finally, in chapter 8 a purification method utilizing perfluorooctanoic acid (PFOA) to solubilize inclusion bodies is presented. This method has a three-fold advantage over conventional solubilization methods because: 1) PFOA can completely solubilize inclusion bodies, 2) PFOA is compatible with Ni-NTA chromatography and 3) PFOA is easily removed by detergent dialysis. Overall, this work represents significant advancements in understanding of the caveolin-1 protein.

Available for download on Wednesday, August 14, 2019

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