Year of Award

2009

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Chemistry

Department or School/College

Department of Chemistry and Biochemistry

Committee Chair

Richard J. Bridges

Commitee Members

Edward Rosenberg, Charles M. Thompson, John M. Gerdes, Kent Sugden, J.B.Alexander Ross

Keywords

EAAT, Glutamate Transporter, Light Inactivation, Mass Spectrometry

Publisher

University of Montana

Abstract

Glia-expressing excitatory amino acid transporter 2 (EAAT2) mediates the bulk of glutamate re-uptake in the human central nervous system (CNS) and is associated with a variety of neurological disorders. Our understanding of the structure and mechanism of this integral membrane protein is limited. The goal of this study was to use pharmacological, mass spectrometric (MS) and photochemical approaches to probe EAAT2. For MS characterization, a hexahis epitope was incorporated into the N-terminus of human EAAT2. The recombinant protein was functionally expressed in HEK 293T cells and purified through a single-step nickel column. In-gel and in-solution trypsin digestions were conducted on the isolated protein. Overall, eighty-nine percent sequence coverage of the protein was achieved. An 88-amino acid tryptic peptide covering the proposed substrate binding site was revealed after N-deglycosylation. This study provided an efficient and simple method to purify, digest and characterize integral membrane proteins by MS. In addition, the EAAT2 peptide fingerprint obtained by digestion offered a template for later protein modification studies. In an effort to design photoaffinity labels for hEAAT2, a series of aryl diaminopropionic acids and aryl aspartylamide compounds were synthesized and characterized as potent EAAT inhibitors. Compounds containing 9-fluorenone groups were found to be able to irreversibly inactivate EAATs under UV-A illumination. The mechanism underlying the photo-inactivation was shown to be singlet oxygen mediated protein oxidation. The specificity of the photo-inactivation was illustrated by the protection effects of inhibitors, as well as the proximity between the transporter and ligands. Trypsin digestion and MS analyses revealed a mass change of a peptide from hEAAT2 binding pocket in the photo-inactivated protein. Molecular docking results supported our speculation that a tryptophan residue was oxidized during the photo-inactivation. The identification of possible EAAT2 photo-inactivation site provided additional information for the location of the EAAT2 lipophilic interaction domain.

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© Copyright 2009 Ran Ye