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Title page for ETD etd-12112008-140102

Type of Document Dissertation
Author Chao, Chih-Kai
URN etd-12112008-140102
Title The vesicular glutamate transporter (VGLUT): heterologous expression, proteoliposome, computational and mass spectral studies
Degree Doctor of Philosophy
Department Department of Biomedical & Pharm. Sciences
Advisory Committee
Advisor Name Title
Charles M Thompson Committee Chair
  • vesicular glutamate transporter
Date of Defense 2008-11-20
Availability unrestricted

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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umi-umt-1113.pdf 3.09 Mb

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