ULTRAFAST TIME-RESOLVED CRYSTALLOGRAPHY ON SCAPHARCA TETRAMERIC HEMOGLOBIN

甲鱼四聚体血红蛋白的超快时间分辨晶体学

• 批准号: 8171968
• 负责人: WILLIAM E ROYER
• 金额: $ 0.55万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171968
• 关键词: Affinity; Binding; Biological Models; Clams; Computer Retrieval of Information on Scientific Projects Database; Crystallography; Data; Funding; Genetic Recombination; Grant; Hemoglobin; Human; Institution; Investigation; Kinetics; Lasers; Ligands; Methods; Oxygen; Pathway interactions; Proteins; Research; Research Personnel; Resources; Source; Structure; System; Time; United States National Institutes of Health; mutant; photolysis; protein function; research study; time use

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 蛤蜊（Scapharca inaequivalvis）拥有两种血红蛋白，它们代表了研究蛋白质变构的特殊模型系统。 两种血红蛋白通过一种与经过深入研究的人类血红蛋白非常不同的结构机制协同结合氧。 二聚血红蛋白，称为 HbI，是具有两个相同亚基的变构的最简单的模型系统。 这种血红蛋白的时间分辨晶体学分析首次提供了实时变构变化的详细结构描述（Knapp 等人 2006，PNAS 103 7649-7654）。 四聚体血红蛋白，称为 HbII，由两个异二聚体形成，每个异二聚体都具有与 HbI 相似的组装。 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和HbII特定突变体的动力学结构途径。 突变体将使我们能够通过改变成对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个亚基的影响。 与 HbI 类似，但与人类血红蛋白不同，我们最近表明，Scapharca HbII 晶体可以在晶体内经历完整的变构转变。 因此，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 CO 配体血红蛋白晶体的激光光解引发。在100皮秒到100微秒的不同时间点，将通过Laue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经历从配体形式到未配体形式的变构转变时的动力学途径。