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

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。 最近从流感嗜血杆菌中产生了Co(II)取代的β-碳酸氢酶。Co(II)酶的可见光谱对酶的变构状态很敏感。这种酶的X射线结构分析对于(1)证明Co(II)酶与野生型锌(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)是直接与金属离子结合，还是与变构中心结合，或两者兼而有之。这种酶的两个样品已经结晶，存在和不存在碳酸氢根离子配体。这些晶体的尺寸为0.2-0.3毫米，是我们去年4月收集的未成功数据的单斜晶型的改进形式。虽然我们还没有筛选这些晶体，但我们预计它们会像我们之前的样品一样衍射(大约。2.0 A) 为了探索变构结合位点在该酶中的作用，制备了流感嗜血杆菌碳酸酐酶变构体R64A。Arg64被认为是与变构部位结合的碳酸氢根离子中的关键残基。这种酶的X射线结构分析对于(1)确定该酶由于突变而采用了两种变构状态中的哪一种，以及(2)重碳酸盐离子是否能与部分修饰的变构结合部位结合是很重要的。这种酶的两个样品已经结晶，有碳酸氢根离子存在和没有碳酸氢根离子存在。晶体大小为0.2-0.4毫米，明显为四方(最有可能是P41212)，类似于我们制备的其他变体的晶体。我们还没有对这些晶体进行筛选，但根据过去的经验，我们预计它们会很好地衍射。 我们总共需要收集4个完整的数据集，上面描述的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异蛋白质结构之一。实验数据的收集和整理应该直截了当。此外，根据时间表的不同，可能会有一名或多名本科生协助收集和分析数据。 有待确定的X射线结构信息将对理解流感嗜血杆菌碳酸酐酶的变构调节机制非常重要，该酶被认为利用了一种独特的金属配体交换机制。更广泛地说，这里确定的结构将增加关于β-碳酸氢酶的越来越多的结构信息体，这将有助于理解这个变构部位可能是如何从它的非变构同系物进化而来的。 2007年4月，我们在CHESS上收集了相关样品和晶体形态的数据。这项工作是我们先前调查的后续工作：Jeff D.Cronk、Roger S.Rowlett、Kam Y.J.Zhang、Tu经匡图、James A.Endrizzi、Joseph Lee、Peter C.Gareiss和Jeffrey R.Preiss，

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. The information resulting from the X-ray structures to be determined will be important to the understanding of the allosteric regulatory mechanism of H. influenzae carbonic anhydrase, which is hypothesized to utilize a unique metal ligand-exchange mechanism. More generally, the structures determined here will add to a growing structural body of information for beta-carbonic anhydrases that will aid understanding about how this allosteric site may have evolved from its non-allosteric homologs. We collected data at CHESS for related samples and crystal forms in April 2007. This work is follows up on our previous investigation: Jeff D. Cronk, Roger S. Rowlett, Kam Y. J. Zhang, Chingkuang Tu, James A. Endrizzi, Joseph Lee, Peter C. Gareiss, and Jeffrey R. Preiss,