SYNTHETIC SYMMETRIZATION TO DETERMINE THE STRUCTURE OF CELA

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 蛋白质结晶仍然是X射线结晶学中的一个主要瓶颈。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体更容易结晶。天然的单体蛋白质可以人工合成同源二聚体。我们的方法是通过在目标蛋白质中引入单半胱氨酸来创建同源二聚体蛋白质，然后将其氧化以在两个单体之间形成二硫键。通过在不同的序列位置引入单个半胱氨酸，人们可以产生各种合成的蛋白质二聚体版本，每个结构都有望展示其自己的结晶行为。在早期的工作中，我们使用T4溶菌酶作为模型系统，展示了该方法的潜在用途。在这里，我们报告了该方法的成功应用于Thermotoga maritima cela，一种与蛋白质数据库中先前报道的具有结构的蛋白质具有低序列同源性的嗜热内切聚糖酶。尽管对结晶条件进行了广泛的调查，但这种蛋白质以其自然单体的形式抵制结晶。

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.