PHYSICO-CHEMICAL STUDIES OF THE VESICULAR GLUTAMATE TRANSPORTER 1 (VGLUT1)
The vesicular glutamate transporter 1 (VGLUT1) is an important membrane protein located in glutamatergic synaptic vesicles. It is responsible for the storage and release of the excitatory neurotransmitter glutamate. VGLUT1 is a highly hydrophobic integral membrane protein with a molecular weight around 61 kD. The tertiary structure of VGLUT1 is still unknown. In our study, recombinant VGLUT1 was expressed in Pichia pastoris and purified using either a nickel chelating column or cobalt-coated Dynabeads. The HiTrapTM nickel chelating column proved to be more efficient in purification of recombinant VGLUT1 than Dynabeads. To study the physico-chemical properties and structure of VGLUT1 and advance our understanding of the membrane topology, FITC was used to modify VGLUT1 in solution. On average, 5.35 ¡À 1.10 lysines were labeled with FITC in each VGLUT1 molecule. Trypsin, endoproteinase Glu-C and Arg-C were used to digest FITC labeled VGLUT1 for mass spectrometry analysis. Mass spectrometry and other proteomics techniques were applied to identify labeled residues. Nine lysine residues were revealed to be labeled by FITC in total, among which 8 lysines (K10, K25, K140, K196, K272, K339, K378, and K507) are from native VGLUT1 and one is located at myc epitope (K569).
Advisor:Charles M. Thompson; Keith Parker; Stephen Sprang
School:The University of Montana
School Location:USA - Montana
Source Type:Master's Thesis
Keywords:department of biomedical pharm sciences
Date of Publication:01/15/2009