DESIGN OF SSPB VARIANTS FOR PROBING SSPB FUNCTION

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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.