ULTRAFAST TIME-RESOLVED CRYSTALLOGRAPHY ON SCAPHARCA TETRAMERIC HEMOGLOBIN

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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。二聚体血红蛋白，称为HBI，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详细结构描述(Knapp et.艾尔2006年，PNAS 103 7649-7654)。四聚体血红蛋白，称为HbII，由两个异二聚体形成，每个异二聚体都有类似于HBI的组装。然而，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链重组动力学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。与HBi类似，但与人类血红蛋白不同，我们最近证明Scapharca HbII晶体可以在晶体内经历完全变构转变。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。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The clam, Scapharca inaequivalvis, possesses two hemoglobins that represent exceptional model system 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 detailed structural description of allosteric changes in real time (Knapp et. al. 2006, PNAS 103 7649-7654). The tetrameric hemoglobin, termed HbII, is formed from two heterodimers, each of which has a similar assembly to that of HbI. However, 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 kinetics, 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.