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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 我们的实验室是NIH蛋白结构倡议的膜蛋白结构生物学联盟的一部分，它正在积极开发技术，以克服在真核跨膜蛋白的结构测定中的重要瓶颈。这包括优化溶解化和结晶条件以提高晶体的衍射质量。我们正在使用SAN（小角度中子散射）和对比变化实验，以确定与纯化的膜蛋白结合的去污剂的空间分布Bor1p，Bor1p，一种来自酿酒酵母的硼酸盐转运蛋白，SLC4超级家族的成员，包括人类元素的重要群体散布，包括人类繁殖的持续时间，是属于人类元素的持续时间。洗涤剂和蛋白质将有可能确定溶液条件变化（包括洗涤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）如何影响BOR1P/洗涤剂分布如何受到溶液条件的变化。 研究该分布如何根据所用洗涤剂以及这种变化如何与蛋白质的流体动力和功能特性相关，这一点非常重要。这些信息可用于制定优化将促进蛋白质结晶的洗涤剂条件选择的策略，该过程当前通过反复试验进行。 在我们最初的无研究研究中，我们发现，相同的BOR1P蛋白质纯化的回旋半径（RG）存在很大差异，这可能是由于制剂的洗涤剂含量的差异很小。在我们下一次在2010年12月在Oak Ridge Laboratories上进行的不在运行之前，我们建议在国际象棋上使用SAXS，以通过分析BOR1P的分析溶液中的溶液中，以识别RG变化的来源，其清洁剂浓度的变化很小。 SAXS提供了一种评估SAN不可能进行的样品的快速方法。 SAXS实验将在一系列的BOR1P/洗涤剂溶液上进行，具有不同的相对量和适当的无蛋白质和无洗涤剂缓冲液控制的溶液。此外，我们有兴趣评估用于研究与不同蛋白质添加剂结合的BOR1P的溶液构象变化的SAX。这些实验的结果将与一组BOR1P TM（熔化温度）测量进行比较，该测量评估了不同添加剂对BOR1P TM的影响。