Date of Award

Spring 5-15-2018

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Biochemistry)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Integral membrane proteins (IMPs) mediate molecular transport and signaling across cellular and organelle membranes, and are essential for many biological processes. Defects in the structure or function of these proteins can lead to a variety of diseases and disorders. Endogenous ligands and therapeutics regulate these proteins by binding to specific, but often unknown sites. This is especially true for hydrophobic ligands and lipids that bind to sites within the transmembrane domains (TMD). Photoaffinity labeling-mass spectrometry (PAL-MS) is a powerful approach to studying sites of protein-ligand interactions, but analysis of membrane proteins by PAL-MS is hampered by technical impediments such as poor solubility, ionization, and fragmentation of photolabeled peptides. In this thesis, I have developed chemical tools and mass spectrometric methods that dramatically improve the identification of ligand binding sites in IMPs. Most notably, by pioneering the use of FLI-tag, I have shifted the focus from fragmentation spectra, which are selectively acquired, to MS1 spectra, which are acquired for every peptide that reaches the mass spectrometer. I have applied these tools to map sterol binding sites in two proteins: VDAC, a beta barrel IMP in the mitochondria, and GLIC, a prokaryotic pentameric ligand gated ion channel. Neurosteroid and cholesterol photolabeled sites were determined in both proteins. Surprisingly, the two sterols occupy the same or overlapping sites, but appear to bind in different orientations. This research suggests that therapeutics or endogenous ligands, such as neurosteroids, may be competing with lipids, such as cholesterol, for their binding and effect.

Language

English (en)

Chair and Committee

Alex S. Evers

Committee Members

Douglas Covey, Michael Gross, Jason Held, Jeanne Nerbonne,

Comments

Permanent URL: https://doi.org/10.7936/K7N8797D

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