DESIGN OF SSPB VARIANTS FOR PROBING SSPB FUNCTION

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。 蛋白质设计是创造用于探测蛋白质功能的新试剂的有用工具。Sauer实验室的Dan Bolon创造了几种设计的SspB适配蛋白变体，他已经用这些变体来测试SspB功能的模型。SspB与设计过程中的功能相关的特征是二聚体界面(SspB形成对称的二聚体)、底物结合裂解和底物结合结构域的c-末端延伸，这些已被证明与ClpX直接相互作用。在一种设计中，半胱氨酸被工程化为SspB及其底物，即ssrA标签，以使适配器及其底物交联。在另一种设计中，SspB的丙氨酸74被突变为谷氨酰胺。这一设计旨在干扰底物结合。第三个设计实验重新设计了SspB二聚体界面，使其不是对称的，从而允许在具有不同底物和/或ClpX结合特性的单体之间形成异二聚体。

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. Protein design is a useful tool for creating novel reagents for probing the function of proteins. Dan Bolon of the Sauer lab has created several designed variants of adaptor protein SspB that he has used to test models of SspB function. The features of SspB related to function that were subjected to the design process were the dimer interface (SspB forms a symmetric dimer), the substrate binding cleft and the c-terminal extensions from the substrate binding domain that have been shown to interact directly with ClpX. In one design, cysteines were engineered into SspB and its substrate, the ssrA tag, in order to crosslink the adaptor and its substrate. In another design, alanine 74 of SspB was mutated to glutamine. This design was intended to interfere with substrate binding. The third design experiment re-engineered the SspB dimer interface so that it would not be symmetric, allowing formation of heterodimers between monomers with different substrate- and/or ClpX-binding properties.