<P>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.</P>
| Identifer | oai:union.ndltd.org:MONTANA/oai:etd.lib.umt.edu:etd-12112008-140102 |
| Date | 15 January 2009 |
| Creators | Chao, Chih-Kai |
| Contributors | Charles M Thompson |
| Publisher | The University of Montana |
| Source Sets | University of Montana Missoula |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lib.umt.edu/theses/available/etd-12112008-140102/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Montana or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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