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.

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。 蛤scapharca inaeequivalvis拥有两个血红蛋白，代表了用于研究蛋白质变构的特殊模型系统。 两种血红蛋白都使用与研究较精确的人类血红蛋白有很大不同的结构机制结合氧气。 称为HBI的二聚体血红蛋白是具有两个相同亚基的变构的最简单模型系统。 该血红蛋白的时间分辨晶体学分析首次提供了实时变构变化的初步结构描述（Knapp等人2006 PNAS 103 7649-7654）。 尽管这些实验的总体成功取得了成功，但主要的缺点是晶体中极高的Geminate重新启动水平，这在变构转变过程中大大降低了信号。 我们对配体迁移的分析包括时间分辨晶体学实验解决方案实验和计算分析（Knapp等，2009年的结构17）强烈表明，通过远端远端远离抗激替氨酸门排出所需的阻尼瞬态亚基旋转，晶体晶格限制了配体出口。 这些实验还揭示了通过“后门”通道的潜在替代出口路线。 我们正在产生突变体，该突变体将使配体在晶格的紧密范围内通过此后门退出。 其中之一已经通过光学实验显示，以减少晶体中的Geminate重新插入。 我们建议将此类突变体与基本升级的生物束线14-IDB一起使用，以显着提高对基于合作氧结合的结构事件进展的理解。 例如，这将使我们能够定义结构过渡的领先和滞后组件，以识别触发以后事件的结构事件。 此外，我们打算使用变构突变体在交替的T和R状态下阐明蛋白质松弛，并剖析变构转变的个体结构成分。 称为HBII的四聚体血红蛋白是由两个异二聚体组成的，每个异二聚体都具有与HBI相似的组装。 两个不同的亚基的存在将允许研究一个亚基如何影响第二个亚基，这在两倍的对称HBI中是不可能的。 因此，我们建议使用时间分辨的X射线衍射实验来阐明四聚体HBII和HBII的特定突变体中的动力学结构途径。 突变体将使我们能够通过更改Geminate重组特性或将一个亚基锁定在高亲和力或低亲和力状态下，将一种亚基类型对第二个亚基的影响分开。 像HBI一样，但与人类血红蛋白不同，我们最近表明Scapharca HBII晶体可以在晶体内进行完整的变构过渡。 结果，该系统非常适合变构蛋白功能的时间分辨晶体学实验。变构转变将由共结合的血红蛋白晶体的激光光解触发。在不同的时间点，从100秒到100微秒的衍射数据不等。在这些时间点获得的结构将揭示动力学途径，因为蛋白质从配体到非配体形式的变构转变。