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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 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。 二聚体血红蛋白称为HBI，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详细结构描述（Knappet。Al。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。 但是，存在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的特异性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重组动力学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。在从100秒到100微秒的各个时间点上，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动力学途径，因为蛋白质从配体到非配体形式的变构转变。