Date

2015

Document Type

Dissertation

Degree

Doctor of Philosophy

Department

Chemistry

First Adviser

Glover, Kerney J.

Other advisers/committee members

Flowers, Robert A.; Lowe-Krentz, Linda; Thevenin, Damien

Abstract

Caveolae are 50-100 nm invaginations in the plasma membrane of eukaryotes that are involved in a number of important cellular processes. The integral membrane protein, caveolin-1, is a key architectural component within caveolae. Despite the preeminent role that caveolin holds in maintaining functional caveolae, there is no three-dimensional structure available. Part of understanding the three-dimensional structure of caveolin requires defining its depth within the biological membrane. Only a hazy picture of caveolins topology exists, it is known that the global topology of the protein is such that the N- and C- termini reside on the cytoplasmic face of the membrane. In this body of work, the topological disposition of the integral membrane protein caveolin-1 reconstituted into model membrane systems is elucidated using biochemical and biophysical techniques. Chapter 1 is provided to give adequate background needed to familiarize the reader with the body of knowledge pertaining to the caveolin protein, vehicles for studying membrane proteins, as well as biophysical techniques that are employed in the remaining chapters. In Chapter 2, the characterization of a major methodological advancement using perfluorooctanoic acid-lipid mixtures to reconstitute protein into liposomes by detergent dialysis is presented. This chapter tackled the challenge of natively refolding caveolin-1 from a highly-purified denatured state and may be useful for reconstitution studies in general. In Chapter 3, a combined biophysical approach using fluorescence spectroscopy, nuclear magnetic resonance, and molecular dynamics simulations is taken to generate a model of caveolin-1 (residues 82-136) within a lipid bilayer. These studies strongly suggested that caveolin contains a membrane embedded turn, an unusual motif in membrane proteins. Chapter 4 deepens and supports the model presented in Chapter 3 by employing cysteine scanning mutagenesis to examine the accessibility of the caveolin-1 scaffolding domain (residues 82-101), a region which is critical to the proteins function. These studies pinpoint the caveolin scaffolding domain as being the region of the protein that first enters the bilayer and provide a clear rationale for how this region can interact with a diverse group of soluble and membrane bound ligands. Chapter 5 examines the topological significance of two major factors thought to impact the caveolin-1 conformation and aqueous exposure, a highly conserved proline residue (P110) located within the putative intramembrane turn region and the inclusion of cholesterol. Near and far ultraviolet circular dichroism measurements paired with single tryptophan mutant λmax and fluorescence quenching experiments provide insight into structural and accessibility changes that the P110A mutation and translocation of the protein into a cholesterol rich environment bring about. Chapter 6 details the synthesis of a novel lipid that contains an indole headgroup (indole-PE) as well as its potential as a molecular ruler in fluorescence studies. Importantly this probe draws light on the behavior of commonly employed tryptophan fluorescence quenching reagents and aids in the interpretation of tryptophan fluorescence quenching experiments performed on caveolin-1 single tryptophan mutants reconstituted into phospholipid bicelles.

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Chemistry Commons

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