Research Project
Grant-in-Aid for Scientific Research (C)
In bacteria, one third of newly synthesized proteins must be translocatedacrossthecytoplasmicmembranetolocalize their final destinations. In this reaction, SecA and SecYE complex have central roles. The former is an essential ATPase to drive the reaction and the latter is an evolutionally conserved membrane protein complex, which is thought to form a specific channel for a translocatingpolypeptide. However, molecular mechanisms of the reaction still remain poorly understood. In addition, bacteria have a multi-path membrane protein, SecDF, which form a translocon-associated complex required for efficient preprotein translocation and membrane protein integration.In this study, we established over-expression in E.coli and purification systems of these Sec factors from Thermusthemophilus HB8, and crystallized their proteins. We succeeded in determination of 3 D structure of TSecA at 2.8A resolution. Contrary to previous reports, the structure revealed occurrence of a parallel dimer. Biochemical studies also confirmed this conclusion and supported that this parallel dimer was dominant in soution. In the case of TSecDF, we obtained complete data sets at 3.7 A resolution and started to build a molecular model of TSecDF. Although we obtained good-shape TSecYE crystals, they only diffracted X-rays to 6 A. To improve the diffraction quality of TSecYE crystals, we prepared monoclonal antibodies against TSecYE and selected one that stably bound to the TSecYE complex. Fab fragments prepared from the monoclonal antibody seem to be a good tool to stabilize the TSecYE complex in crystal lattice.
All 2006 2005 2004
All Journal Article (9 results)
Acta Crystallographica Section F. 62
Pages: 376-380
Acta Ciystallographica Section F 62
Acta Crystallographica Section F 62
Genes & Development 19
Pages: 436-444
Genes and Development 19
J. Bacteriol. 186
Pages: 3960-3969
J.Bacteriol. 186
Journal of Bacteriology 186
