DESIGN OF SSPB VARIANTS FOR PROBING SSPB FUNCTION

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定为研究者机构。蛋白质设计是一个有用的工具，创造新的试剂，探测蛋白质的功能。Sauer实验室的Dan Bolon创造了几种设计的衔接蛋白SspB变体，他用这些变体来测试SspB功能的模型。与经历设计过程的功能相关的SspB的特征是二聚体界面（SspB形成对称二聚体）、底物结合裂缝和来自底物结合结构域的C-末端延伸，其已被证明与ClpX直接相互作用。在一种设计中，将半胱氨酸工程化到SspB及其底物ssrA标签中，以便交联衔接子及其底物。在另一个设计中，SspB的丙氨酸74突变为谷氨酰胺。该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，使其不对称，允许在具有不同底物和/或ClpX结合特性的单体之间形成异源二聚体。

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.