Crystallization and preliminary X-ray analysis of the bacterial membrane transporters

Department of Bioscience, School of Bioscience and Biotechnology, Tokyo Institute of Technology* Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Japan**
â—‹Yoshiki Tanaka* Motoyuki Hattori** Shuya Fukai** Ryuichiro Ishitani** Osamu Nureki**

Membrane transporters have critical physiological roles in all living organ isms. To survive and function properly, cells exchange various transport sub strates, such as amino acids, sugars and metal ions with their environment, which is performed by membrane transporters embedded in the cell membrane. A pproximately 10 percent of bacterial genomes consist of membrane transporters. Although the structural information is essential for understanding the functional mechanism of membrane transporters, the number of their available high-resolution structure is limited, because membrane protein structure is very difficult to be determined. To determine the novel structure of membrane transporters, we have cloned the several membrane transporters from thermophilic bacteria. The membrane transporters were overexpressed in E. coli, purified, and tried to be crystallized. In the initial crystallization screening, a crystallization robot enabled us to perform high-throughput screening, despite the limited amount of purified transporter proteins. Until now, we have succeeded in crystallization of two full-length and one cytosolic domain of the membrane transporters. The native and selenomethionine-substituted crystals of the cytosolic domain diffracted to 2.1 and 2.2 angstrom resolution, respectively, using synchrotron radiation. The structure determination of the cytosolic domain by multi-wavelength anomalous dispersion method is now in progress. Moreover, we are developing its transport assay in the liposome
- reconstituted system.