Lawrence Berkeley National Laboratory

An important aim of the Genomics GTL Protein Complex Analysis Project (PCAP) is the isolation and identification of membrane protein complexes from D. vulgaris. The cataloging of complex subunit constituents from this organism, grown under normal and stressed conditions, will support the long-range goal of modeling stress responses in D. vulgaris relevant to the detoxification of metal and radionuclide contaminated sites. Isolation of endogenous D. vulgaris membrane protein complexes in quantities sufficient for chromatographic analysis requires substantial amounts of cell membranes. Twenty liter cultures of D. vulgaris typically yielded about 50 milligrams of total membrane proteins. To extract inner and outer membrane protein complexes stably and with maximal yield, we have used a multi-step procedure in which the bacterial membrane is sequentially processed. Mernbranes were initially treated with a mild detergent to extract proteins primarily from the inner membrane. The residual membrane pellet, enriched with proteins of the outer membrane, was solubilized using a more aggressive detergent. Chromatographic procedures found effective in purifying complexes involved the use of ion exchange, hydroxyapatite and molecular sieve media. SDS-PAGE of the various chromatographic fractions was used to tentatively identify subunits of complexes based on a display of co-elution. Molecular sieve fractions were subjected to native PAGE to further isolate potential complexes. Proteins of the native gel bands, in tum, were extracted and run on SDSPAGE. These gels revealed whether a putative complex was indeed composed of lower molecular weight subunits, and provided samples well suited for in-gel processing and mass spectrometry analysis. In this manner, a number of homo-and heteromeric complexes, ranging in weight from about 70 to 400 kDa, have been identified in this first year. Use of a free-flow electrophoresis device being developed in our group in combination with larger-scale processing of cell membranes is expected to significantly improve the isolation and identification of complexes over the next project year.