STRUC DETERMINATION OF METAL-SUBSTITUTED & ALLOSTERIC SITE VARIANTS OF H INFLU

金属取代物的结构测定

• 批准号: 7955561
• 负责人: Roger Scott Rowlett
• 金额: $ 1.11万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7955561
• 关键词: Adopted; Allosteric Site; Bicarbonate Ion; Bicarbonate Ions; Binding; Binding Sites; Computer Retrieval of Information on Scientific Projects Database; Data; Data Collection; Data Set; Enzymes; Funding; Grant; Haemophilus influenzae; Institution; Ions; Ligands; Metals; Molecular; Mutation; Proteins; Research; Research Personnel; Resources; Roentgen Rays; Role; Sampling; Schedule; Source; Structure; Students; United States National Institutes of Health; Variant; base; carbonate dehydratase; experience; improved; protein structure

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. Co(II)-substituted beta-carbonic anhydrase from H. influenzae has recently been produced. The visible spectrum of the Co(II) enzyme is sensitive to allosteric state of the enzyme. X-ray structural analysis of this enzyme is important to (1) demonstrate that the Co(II) enzyme is isostructural with the wild type, Zn(II) enzyme, and (2) determine if bicarbonate ion (both a substrate and allosteric effector) binds directly to the metal ion, to the allosteric site, or both. Two samples of this enzyme have been crystallized, with and without bicarbonate ion ligand present. The crystals, which are 0.2-0.3 mm in size, are an improved form of monoclinic crystals we collected unsuccessful data on last April. While we have not yet screened these crystals, we would expect them to diffract as well as our prior sample (approx. 2.0 A) Variant R64A of H. influenzae carbonic anhydrase has been prepared to explore the role of the allosteric binding site in this enzyme. Arg64 is believed to be a critical residue in bicarbonate ion binding to the allosteric site. X-ray structural analysis of this enzyme is important to (1) determine which of the two allosteric states the enzyme has adopted as a result of this mutation, and (2) whether or not bicarbonate ion can bind to the partially modified allosteric binding site. Two samples of this enzyme have been crystallized, with and without bicarbonate ion present. The crystals are 0.2-0.4 mm in size and clearly tetragonal (most likely P41212) , similar to crystals of other variants we have prepared. We have not yet screened these crystals, but expect them to diffract well based on past experience. Altogether we need to collect 4 complete datasets, two for each protein sample described above. Structures will be solved by molecular replacement, using the wild-type enzyme or one of our existing variant protein structures. The experimental data collection and reduction should be straightforward. In addition, there is the possibility that one or more undergraduate students could assist in data collection and analysis, depending on scheduling.

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH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 中心，不一定是研究者的机构。 Co（II）-取代的β-碳酸酐酶。流感病毒是最近生产的。Co（II）酶的可见光谱对酶的变构状态敏感。这种酶的X射线结构分析对于（1）证明Co（II）酶与野生型Zn（II）酶是同构的，以及（2）确定碳酸氢根离子（既是底物又是变构效应物）是否直接与金属离子、变构位点或两者结合是重要的。该酶的两个样品已经结晶，存在和不存在碳酸氢根离子配体。晶体大小为0.2-0.3毫米，是我们去年4月收集的不成功数据的单斜晶体的改进形式。虽然我们还没有筛选这些晶体，但我们希望它们能像我们之前的样品一样表现出色（约为100%）。2.0 A）、 H.制备了流感病毒碳酸酐酶，以探索该酶中变构结合位点的作用。Arg 64被认为是碳酸氢根离子与变构位点结合的关键残基。 该酶的X射线结构分析对于（1）确定该酶由于该突变而采用了两种变构状态中的哪一种，以及（2）碳酸氢根离子是否可以结合到部分修饰的变构结合位点是重要的。该酶的两个样品已经结晶，存在和不存在碳酸氢根离子。晶体的大小为0.2-0.4 mm，并且明显是四聚体（最有可能是P41212），类似于我们制备的其他变体的晶体。我们还没有筛选这些晶体，但根据过去的经验，希望它们能很好地发挥作用。 我们总共需要收集4个完整的数据集，每个蛋白质样本两个。结构将通过分子置换来解决，使用野生型酶或我们现有的变体蛋白质结构之一。实验数据的收集和简化应该是直接的。此外，有一个或多个本科生可以协助数据收集和分析的可能性，这取决于时间安排。