OLIGOMERIZATION STATE DETERGENT-ASSOCIATED BORON TRANSPORT MEMBRANE PROT BOR1P

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Our laboratory, which is part of the Membrane Protein Structural Biology Consortium of the NIH Protein Structure Initiative, is actively developing technologies to overcome significant bottlenecks in the structure determination of eukaryotic transmembrane proteins. This includes the optimization of solubilization and crystallization conditions to improve the diffraction quality of crystals. We are using SANS (small angle neutron scattering) with contrast variation experiments to determine the spatial distribution of detergent bound to a purified membrane protein, Bor1p, a borate transporter from S. cerevisiae and member of the SLC4 superfamily, an important group of membrane proteins that includes the human anion transporters, AE1, 2, and 3. Because of the inherently different scattering length densities of the detergents and protein it will be possible to determine how the Bor1p/detergent distribution is affected by changes in solution conditions, including changes in detergent type and concentration, and the presence of inhibitors and substrates bound to Bor1p. It is of particular importance to study how this distribution changes depending on the detergent used and how such changes correlate with hydrodynamic and functional properties of the protein. This information can be used to develop strategies for optimizing the choice of detergent conditions that will promote protein crystallization, a process that currently is performed by trial and error. In our initial SANS studies we have found that there is considerable variation in the radius of gyration (Rg) of identical protein purifications of Bor1p, which may result from small differences in the detergent content of the preparations. Prior to our next SANS run at Oak Ridge Laboratories in December 2010, we propose to use SAXS at CHESS to help in identifying the source of the Rg variation through analysis of Bor1p in solution with small variations in detergent concentration. SAXS provides a rapid method of evaluating samples not possible with SANS. The SAXS experiments will be performed on a series of Bor1p/detergent solutions with differing relative amounts of detergent and with the appropriate no protein and no detergent buffer controls. Additionally, we are interested in evaluating SAXS for studying solution conformational changes of Bor1p bound to different protein additives. The results from these experiments will be compared to a set of Bor1p Tm (melting temperatures) measurements that evaluated the effect of different additives on Bor1p Tm.