Year of Award

2008

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Pharmaceutical Sciences

Other Degree Name/Area of Focus

Pharmacology

Department or School/College

College of Health Professions and Biomedical Sciences

Committee Chair

Charles M. Thompson

Commitee Members

Mark L. Grimes, Diana I. Lurie, Keith K. Parker, David J. Poulsen

Keywords

vesicular glutamate transporter

Abstract

Vesicular glutamate transporters (VGLUTs) are integral membrane proteins that uptake glutamate into synaptic vesicles and are involved in glutamatergic neurotransmission. Since VGLUTs were identified and cloned, efforts have been made to characterize their functional roles. However, due to experimental limitations, the structural features of VGLUT protein remain unclear. In an attempt to better understand VGLUTs, computational and biochemical approaches were employed to characterize them. Plasmid DNA encoding rat VGLUT1 was constructed, amplified and expressed in Pichia pastoris to produce VGLUT1 protein. Immobilized metal affinity chromatography (IMAC) was employed to purify the protein for structural analysis by mass spectrometry and to develop a functional transporting system, VGLUT1 proteoliposomes. Transmembrane topology and homology models of VGLUT1 were generated by web-based and in-house programs. The computational analysis implies that VGLUT1 protein appears to have 12-transmembrane domains. Chemical and enzymatic cleavages and mass spectral analysis of denatured and proteoliposome-reconstituted VGLUT1 protein show that the experimental results are consistent with the computational models. These results provide basic insight into VGLUT protein structure for neuropharmacology studies related to glutamatergic neurotransmission.

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© Copyright 2008 Chih-Kai Chao