OLIGOMERIZATION STATE DETERGENT-ASSOCIATED BORON TRANSPORT MEMBRANE PROT BOR1P

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 我们的实验室是NIH蛋白质结构倡议的膜蛋白结构生物学联盟的一部分，正在积极开发技术，以克服真核生物跨膜蛋白结构测定中的重大瓶颈。这包括优化增溶和结晶条件，以提高晶体的衍射质量。我们正在使用SANS（小角中子散射）与对比度变化实验，以确定空间分布的洗涤剂结合到一个纯化的膜蛋白，Bor 1 p，硼酸盐转运蛋白从S。酿酒酵母和SLC 4超家族成员，SLC 4超家族是一组重要的膜蛋白，包括人阴离子转运蛋白AE 1、2和3。由于去污剂和蛋白质的散射长度密度固有不同，因此可以确定Bor 1 p/去污剂分布如何受到溶液条件变化的影响，包括去污剂类型和浓度的变化，以及与Bor 1 p结合的抑制剂和底物的存在。 特别重要的是研究这种分布如何根据所使用的洗涤剂而变化，以及这种变化如何与蛋白质的流体动力学和功能特性相关。这些信息可用于开发优化洗涤剂条件选择的策略，以促进蛋白质结晶，这是一个目前通过试验和错误进行的过程。 在我们最初的SANS研究中，我们发现Bor 1 p的相同蛋白纯化的回转半径（Rg）存在相当大的变化，这可能是由于制剂中去污剂含量的微小差异。在2010年12月我们在橡树岭实验室进行下一次SANS运行之前，我们建议在CHESS使用SAXS，通过分析溶液中的Bor 1 p（洗涤剂浓度存在微小变化）来帮助确定Rg变化的来源。SAXS提供了SANS无法实现的快速样品评价方法。SAXS实验将在一系列Bor 1 p/去污剂溶液中进行，这些溶液含有不同相对量的去污剂以及适当的无蛋白和无去污剂缓冲液对照。此外，我们有兴趣在评估SAXS研究解决方案的构象变化Bor 1 p绑定到不同的蛋白质添加剂。将这些实验的结果与一组Bor 1 p Tm（熔融温度）测量值进行比较，以评估不同添加剂对Bor 1 p Tm的影响。

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.