OLIGOMERIZATION STATE DETERGENT-ASSOCIATED BORON TRANSPORT MEMBRANE PROT BOR1P

低聚态洗涤剂相关硼传输膜 PROT BOR1P

• 批准号: 8363558
• 负责人: MARK E. DUMONT
• 金额: $ 1.24万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363558
• 关键词: Affect; Anions; Binding; Borates; Boron; Buffers; Crystallization; Detergents; Funding; Grant; Human; Integral Membrane Protein; Laboratories; Length; Measurement; Membrane Proteins; National Center for Research Resources; Neutrons; Preparation; Principal Investigator; Process; Property; Protein Structure Initiative; Proteins; Radial; Relative (related person); Research; Research Infrastructure; Resources; Running; Saccharomyces cerevisiae; Sampling; Series; Solutions; Source; Spatial Distribution; Structure; Technology; Temperature; Transmembrane Transport; United States National Institutes of Health; Variant; cost; density; improved; inhibitor/antagonist; interest; melting; member; protein purification; rapid technique; research study; structural biology

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.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的首席研究员可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 我们的实验室是 NIH 蛋白质结构计划膜蛋白质结构生物学联盟的一部分，正在积极开发技术以克服真核跨膜蛋白质结构测定中的重大瓶颈。这包括优化溶解和结晶条件以提高晶体的衍射质量。我们使用 SANS（小角中子散射）和对比变化实验来确定与纯化膜蛋白 Bor1p 结合的去污剂的空间分布，Bor1p 是来自酿酒酵母的硼酸盐转运蛋白，也是 SLC4 超家族的成员，SLC4 超家族是一组重要的膜蛋白其中包括人类阴离子转运蛋白 AE1、2 和 3。由于去垢剂和蛋白质的散射长度密度本质上不同，因此可以确定 Bor1p/去垢剂的分布情况受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 Bor1p 结合的抑制剂和底物的存在。 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是特别重要的。该信息可用于制定优化洗涤剂条件选择的策略，以促进蛋白质结晶，目前这一过程是通过反复试验来执行的。 在我们最初的 SANS 研究中，我们发现 Bor1p 的相同蛋白质纯化的回转半径 (Rg) 存在相当大的差异，这可能是由于制备物的去污剂含量存在微小差异所致。在 2010 年 12 月在橡树岭实验室运行下一次 SANS 之前，我们建议在 CHESS 中使用 SAXS，通过分析洗涤剂浓度变化较小的溶液中的 Bor1p 来帮助确定 Rg 变化的来源。 SAXS 提供了一种使用 SANS 无法实现的快速评估样品的方法。 SAXS 实验将在一系列具有不同相对量的去污剂以及适当的无蛋白质和无去污剂缓冲液对照的 Bor1p/去污剂溶液上进行。此外，我们有兴趣评估 SAXS 以研究 Bor1p 与不同蛋白质添加剂结合的溶液构象变化。这些实验的结果将与一组 Bor1p Tm（熔化温度）测量结果进行比较，以评估不同添加剂对 Bor1p Tm 的影响。