Identification and Characterization of Halobacterium species NRC-1 gas vesicle proteins by tandem mass spectrometry

• Main Authors: Meng-Chieh Chen, 陳夢婕
• Other Authors: Wailap Victor Ng
• Format: Others
• Language: en_US
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/26312416756655512994

碩士 === 國立陽明大學 === 醫學生物技術研究所 === 95 === The gas vesicle (GV) of Halobacterium sp. NRC-1 is an intracellular organelle that provides buoyancy to the cell. The gas-filled vesicle envelop is composed of proteins. Here we report the analysis of gas vesicle structure components and the possible gas vesicle biogenesis pathway via protein-protein interaction analysis. In this work, we used either the microwave-assisted HCl partial hydrolysis or trypsin digestion to fragment GV to produce the peptides for mass spectrometry. A total of 1775 partial HCl hydrolysis generated and 70 tryptic GV peptides were identified with a peptide identification (Peptideprophet) probability (p) above 0.9. The identified Gvp proteins including the lemon-shaped GV structure proteins GvpA1, C1, N1, F1, and J1 encoded by gvp-1 gene cluster and the cylindrical-shaped GV proteins GvpA2, H2, J2 encoded by gvp-2 gene cluster. Among these Gvp, GvpN1, H2 and J2 are newly identified structure proteins in purified gas vesicles. We also found that HCl partial hydrolysis is an excellent method to produce peptides that allow us to distinguish GvpA1 and A2. These two similar small GV proteins differ by only 5 amino acids in 3 locations. Furthermore, in order to clarify the gas vesicles biogenesis pathway, each Gvp protein is tagged with a protein A at C-terminus and expressed in NRC-1 to pull down its interacting proteins for mass spectrometric analysis. Most of the identified structural Gvp proteins including GvpC1, N1, H1, F1, and L1 were interacted with GvpA1, the gas vesicle major component. Among these, GvpN1 interacted with GvpI1 suggested that the role of GvpN1 and GvpI1 were enhancers for shaping GVs. Taken together of the above results and previous studies, a better picture of the NRC-1 gas vesicle structure components and gas vesicles biogenesis pathway have been obtained.