SYNTHETIC SYMMETRIZATION TO DETERMINE THE STRUCTURE OF CELA

综合对称确定 CELA 结构

• 批准号: 8361685
• 负责人: TODD O YEATES
• 金额: $ 0.48万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361685
• 关键词: Behavior; Biological Models; Complex; Crystallization; Cysteine; Databases; Enzymes; Funding; Grant; Methods; Muramidase; National Center for Research Resources; Positioning Attribute; Principal Investigator; Proteins; Reporting; Research; Research Infrastructure; Resources; Source; Structure; Surveys; Thermotoga maritima; United States National Institutes of Health; Work; X-Ray Crystallography; cellulase; cost; disulfide bond; interest; monomer; structural biology

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Protein crystallization continues to be a major bottleneck in X-ray crystallography. Previous studies suggest that symmetric proteins, such as homodimers, might crystallize more readily than monomeric proteins or asymmetric complexes. Proteins that are naturally monomeric can be made homodimeric artificially. Our approach is to create homodimeric proteins by introducing single cysteines into the protein of interest, which are then oxidized to form a disulfide bond between the two monomers. By introducing the single cysteine at different sequence positions, one can produce a variety of synthetically dimerized versions of a protein, with each construct expected toexhibit its own crystallization behavior. In earlier work, we demonstrated the potential utility of the approach using T4 lysozyme as a model system. Here we report the successful application of the method to Thermotoga maritima CelA, a thermophilic endoglucanase enzyme with low sequence identity to proteins with structures previously reported in the Protein Data Bank. This protein had resisted crystallization in its natural monomeric form, despite a broad survey of crystallization conditions.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 蛋白质结晶仍然是X射线晶体学的主要瓶颈。以前的研究表明，对称蛋白质，如同源二聚体，可能比单体蛋白质或不对称复合物更容易结晶。天然单体的蛋白质可以人工合成为同型二聚体。我们的方法是通过将单个半胱氨酸引入感兴趣的蛋白质中，然后将其氧化以在两个单体之间形成二硫键来产生同源二聚体蛋白质。通过在不同的序列位置引入单个半胱氨酸，人们可以产生多种合成的二聚体形式的蛋白质，每个构建体都有望表现出自己的结晶行为。在早期的工作中，我们证明了使用T4溶菌酶作为模型系统的方法的潜在效用。在这里，我们报告的成功应用的方法Thermotoga maritima CelA，嗜热内切葡聚糖酶与低序列同一性的蛋白质与以前报道的蛋白质数据库中的结构。尽管对结晶条件进行了广泛的调查，但这种蛋白质以其天然单体形式抵抗结晶。