FREEZE-QUENCH STUDY ON PROTEIN CONFORMATION STATE

蛋白质构象状态的冷冻淬灭研究

• 批准号: 8364073
• 负责人: ELKA R GEORGIEVA
• 金额: $ 4.49万
• 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: 2011-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8364073
• 关键词: Complex; Freedom; Freezing; Funding; Goals; Grant; Label; Modeling; Molecular Conformation; National Center for Research Resources; Paper; Pattern; Principal Investigator; Protein Conformation; Proteins; Research; Research Infrastructure; Resources; Roentgen Rays; Sampling; Solutions; Solvents; Source; Spin Labels; Structure; Techniques; Technology; Temperature; Testing; United States National Institutes of Health; X-Ray Crystallography; alpha helix; chemical bond; cold temperature; conformer; cost; cryogenics; flexibility; protein complex; protein folding; retinal rods

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. PDS conducted on spin-labeled proteins at low temperatures yields distance distributions. However, it is not well understood to what extent protein conformations represented by these distributions correspond to conformations in solution. In general there is an argument that the structure determined at such conditions may be a poor model for highly dynamic (flexible) proteins. Similar arguments, such as protein confinement by the crystal lattice and using cryogenic temperatures for collecting diffraction pattern could be (and are) applied to X-ray crystallography. But we do know that there is often significant correspondence between X-ray and NMR derived structures. Therefore it is important to test what the distance distributions from PDS conducted on proteins in solution in the absence of confinement by crystal contacts may represent. One argument is that the range of conformations given by the distribution of end-to-end distances depends mainly on spring constants and the number of connecting chemical bonds. One would expect to find this for example for a rod like organic molecule or long alpha-helix, however one would need to take a sufficiently long helix in order be able determine the extent of its flexibility and may also need to use conformationally-persistent spin-labels, such as TOAC. This was the approach used in our collaborative paper with D. Budil et al. However, for a folded protein or a protein complex the picture may be more complex, since many bonds allow for substantial freedom and may produce an ensemble of conformations that exchange slow and can get trapped. Therefore, sufficiently rapid freezing that brings the solvent into the glassy state as a matter of microseconds is expected to preserve the ensemble of such conformers, but may depopulate those that exchange with faster rates in microsecond range. We are going to apply freeze-quenching technique, developed in Scholes lab, using different freezing rates on doubly labeled model proteins and to compare distances distributions obtained by PDS with the goals to delineate conformational exchange rates and to test the relevance of distance distributions from PDS to the range of conformations sampled by a protein in solution. .

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 并且子项目的主要研究者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。 在低温下对自旋标记蛋白质进行 PDS 可以得到距离分布。然而，尚不清楚这些分布所代表的蛋白质构象在多大程度上对应于溶液中的构象。一般来说，有一种观点认为，在这种条件下确定的结构对于高度动态（灵活）的蛋白质来说可能是一个糟糕的模型。类似的论点，例如晶格对蛋白质的限制以及使用低温收集衍射图样，可以（并且已经）应用于 X 射线晶体学。但我们确实知道，X 射线和核磁共振衍生的结构之间通常存在显着的对应关系。 因此，测试在没有晶体接触限制的情况下对溶液中的蛋白质进行的 PDS 的距离分布可能代表什么是很重要的。一种论点是，由端到端距离分布给出的构象范围主要取决于弹簧常数和连接化学键的数量。人们期望找到这一点，例如棒状有机分子或长α螺旋，然而，人们需要采用足够长的螺旋才能确定其柔性程度，并且可能还需要使用构象持久的自旋标记，例如TOAC。这是我们与 D. Budil 等人的合作论文中使用的方法。然而，对于折叠蛋白质或蛋白质复合物来说，情况可能更加复杂，因为许多键允许很大的自由度，并且可能产生交换缓慢且可能被捕获的构象集合。因此，足够快的冷冻使溶剂在几微秒内进入玻璃态预计可以保留此类构象异构体的整体，但可能会减少那些在微秒范围内以更快速率交换的构象异构体。 我们将应用 Scholes 实验室开发的冷冻淬灭技术，对双标记模型蛋白质使用不同的冷冻速率，并比较 PDS 获得的距离分布，目的是描绘构象交换率并测试 PDS 距离分布与溶液中蛋白质采样的构象范围的相关性。 。