ULTRAFAST TIME-RESOLVED CRYSTALLOGRAPHY ON SCAPHARCA DIMERIC AND TETRAMERIC H

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 蛤Scapharca inaequalivis拥有两个血红蛋白，代表特殊的模型系统的研究蛋白质变构。 这两种血红蛋白结合氧合作使用的结构机制是非常不同的，从更好地研究人类血红蛋白。 被称为HbI的二聚血红蛋白是具有两个相同亚基的变构的最简单的可能模型系统。 这种血红蛋白的时间分辨晶体学分析首次提供了真实的时间变构变化的初步结构描述（克纳普等）。2006 PNAS 103 7649 - 7654）。 尽管这些实验总体上是成功的，但一个主要的缺点是晶体中非常高水平的成对再结合，这大大降低了变构转变期间的信号。 我们对配体迁移的分析，包括时间分辨晶体学实验、溶液实验和计算分析（克纳普et al. 2009 Structure 17 in press），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退出。 这些实验还揭示了通过"后门"渠道的潜在替代退出途径。 我们正在制造突变体，它将允许配体在晶格的严格限制内通过这个后门离开。 光学实验已经证明，其中一种方法可以减少晶体中的孪晶再结合。 我们建议使用这样的突变体与大幅升级的BioCARS光束线14-IDB，以获得显着改善的理解的基础合作氧结合的结构事件的进展。 例如，这将使我们能够定义结构转变的领先和滞后组件，以识别触发后续事件的结构事件。 此外，我们打算使用变构突变体来阐明蛋白质松弛交替的T和R状态，并剖析个别结构组件的变构转换。 被称为HbII的四聚体血红蛋白由两个异二聚体形成，每个异二聚体具有与HbI相似的组装。 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的X-射线衍射实验来阐明动力学结构途径的四聚体HbII和特定的突变体的HbII。 突变体将允许我们通过改变成对重组特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出一种亚基类型对第二种亚基的影响。 像HbI，但不像人类血红蛋白，我们最近表明，Scapharca HbII晶体可以进行晶体内的完全变构转变。 因此，该系统非常适合于变构蛋白质功能的时间分辨晶体学实验。激光光解CO配体血红蛋白晶体将引发变构转变。在100皮秒到100微秒的不同时间点，将通过劳厄方法收集衍射数据。在这些时间点获得的结构将揭示蛋白质从配体形式到非配体形式的变构转变的动力学途径。

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.