ULTRAFAST TIME-RESOLVED CRYSTALLOGRAPHY ON SCAPHARCA DIMERIC AND TETRAMERIC H

鱼蚶二聚体和四聚体 H 的超快时间分辨晶体学

• 批准号: 8363704
• 负责人: WILLIAM E ROYER
• 金额: $ 2.43万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363704
• 关键词: Affinity; Back; Binding; Biological Models; Clams; Computer Analysis; Crystallography; Data; Distal; Event; Funding; Genetic Recombination; Grant; Hemoglobin; Histidine; Human; Individual; Investigation; Kinetics; Lasers; Ligands; Methods; National Center for Research Resources; Optics; Oxygen; Pathway interactions; Principal Investigator; Property; Proteins; Relaxation; Research; Research Infrastructure; Resources; Rotation; Route; Signal Transduction; Solutions; Source; Structure; System; Time; United States National Institutes of Health; beamline; cost; improved; migration; mutant; photolysis; protein function; research study; structural biology; success; time use

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. The clam Scapharca inaequivalvis possesses two hemoglobins that represent exceptional model systems for the investigation of protein allostery. Both hemoglobins bind oxygen cooperatively using a structural mechanism that is very different from the more well studied human hemoglobin. The dimeric hemoglobin termed HbI is the simplest possible model system for allostery with two identical subunits. Time-resolved crystallographic analysis of this hemoglobin provided for the first time a preliminary structural description of allosteric changes in real time (Knapp et. al. 2006 PNAS 103 7649-7654). Despite the overall success of these experiments a major drawback was the very high level of geminate rebinding in the crystal which substantially reduced the signal during the allosteric transition. Our analysis of ligand migration including time-resolved crystallographic experiments solution experiments and computational analysis (Knapp et al. 2009 Structure 17 in press) strongly suggests the crystal lattice restricts ligand exit by damping transient subunit rotations that are required for exit through a distal histidine gate. These experiments also revealed a potential alternate exit route through a "back door" channel. We are producing mutants that will allow ligands to exit through this back door within the tight confines of the crystal lattice. One of these has already been shown by optical experiments to reduce geminate rebinding in crystals. We propose to use such mutants with the substantially upgraded BioCARS beamline 14-IDB to obtain significantly improved understanding of the progression of structural events that underlie cooperative oxygen binding. This will for instance allow us to define leading and lagging components of the structural transitions to identify those structural events that trigger later events. Moreover we intend to use allosteric mutants to elucidate protein relaxation in alternate T and R states and to dissect individual structural components of the allosteric transition. The tetrameric hemoglobin termed HbII is formed from two heterodimers each of which has a similar assembly to that of HbI. The presence of two different subunits will permit investigation of how one subunit impacts a second subunit which is not possible in the two-fold symmetric HbI. Therefore we propose to use time-resolved x-ray diffraction experiments to elucidate the kinetic structural pathway in the tetrameric HbII and specific mutants of HbII. Mutants will allow us to separate out the effects of one subunit type on the second subunit either by altering the geminate recombination properties or by locking one subunit in a high affinity or low affinity state. Like HbI but unlike human hemoglobin we have recently shown that Scapharca HbII crystals can undergo the full allosteric transition within crystals. As a result this system is well suited for time-resolved crystallographic experiments of allosteric protein function. Allosteric transition will be triggered by laser photolysis of CO-liganded hemoglobin crystals. At various time points ranging from 100 picoseconds to 100 microseconds diffraction data will be collected by Laue methods. The structures obtained at these time points will reveal the kinetic pathways as the protein undergoes its allosteric transition from the liganded to the unliganded form.

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 文蛤(Scapharca Inaequvalvis)拥有两种血红蛋白，它们代表了蛋白质变构研究的特殊模式系统。这两种血红蛋白通过一种与研究更充分的人类血红蛋白非常不同的结构机制协同结合氧气。 称为HBI的二聚体血红蛋白是具有两个相同亚基的变构最简单的可能的模型系统。这种血红蛋白的时间分辨结晶学分析首次实时提供了变构变化的初步结构描述(Knapp et.艾尔2006年PNA 103 7649-7654)。尽管这些实验总体上取得了成功，但一个主要缺点是晶体中的双酯重新结合水平非常高，这在变构转变期间大大减少了信号。我们对配体迁移的分析包括时间分辨结晶学实验、溶液实验和计算分析(Knapp等人)。按下的2009年结构17)强烈表明，晶格通过抑制通过远端组氨酸门退出所需的瞬时亚基旋转来限制配体的退出。这些实验还揭示了一条潜在的替代出口路线--通过“后门”渠道。我们正在生产突变体，它将允许配体在晶格的严格限制内通过这个后门退出。其中之一已经被光学实验证明，以减少晶体中的双晶重新结合。我们建议将这些突变体与大幅升级的BioCARS光束线14-IDB一起使用，以获得对合作氧结合基础上的结构事件进展的显著改善的理解。例如，这将使我们能够定义结构转变的领先和滞后成分，以确定那些触发后来事件的结构性事件。此外，我们打算使用变构突变体来阐明T和R交替状态下的蛋白质松弛，并剖析变构转换的个别结构成分。 称为HbII的四聚体血红蛋白是由两个异二聚体形成的，每个异二聚体的组装都与HBI相似。两个不同亚基的存在将允许研究一个亚基如何影响第二个亚基，这在双重对称HBI中是不可能的。因此，我们建议使用时间分辨X射线衍射实验来阐明四聚体HbII及其特定突变体中的动力学结构途径。突变体将允许我们通过改变双态重组属性或通过将一个亚基锁定在高亲和力或低亲和力状态来分离一种亚基类型对第二亚基的影响。与Hbi类似，但与人类血红蛋白不同，我们最近发现Scapharca HbII晶体可以在晶体内经历完整的变构转变。因此，该系统非常适合于变构蛋白功能的时间分辨结晶学实验。共配体血红蛋白晶体的激光光解将触发变构转变。在从100皮秒到100微秒的不同时间点，将用劳厄方法收集衍射数据。在这些时间点获得的结构将揭示当蛋白质经历从连接形式到非连接形式的变构转变时的动力学路径。