STRINGENT STARVATION PROTEIN A: EXPRESSION, PURIFICATION, CHARACTERIZATION, AND PRELIMINARY X-RAY DIFFRACTION STUDIES

Abstract

Orthologous proteins can be beneficial to crystallographic studies when a protein from an organism of choice fails to crystallize or the protein crystals fail to diffract, because orthologs can replace each other in crystallization trials. The protein sequences will differ enough that the orthologous proteins may crystallize under alternative conditions and diffract to higher resolution. Yet, they are similar enough that they will have the same folding pattern and structure. The use of this multigenomic approach has benefited the crystallization of the transcription factor Stringent Starvation Protein A (SspA). SspA from Escherichia colt was crystallized, however, the crystals failed to diffract well enough for structure determination. Therefore, SspA proteins from Yersinia pestis,Vibrio cholerae, and Pseudomonas aeruginosa were cloned, expressed, purified, and subjected to crystallization trials. The V. cholerae SspA protein failed to crystallize under any conditions tested and the P. aeruginosa SspA protein did not form crystals that were suitable for data collection. On the other hand, Y pestis SspA crystallized readily and the crystals diffracted to 2.0 A. The crystal structure will be determined and used to construct models of the structures of the E. coil, V. cholerae, and P. aeruginosa SspA proteins. The structure of SspA will help to illuminate its relationship with RNA polymerase and other transcription factors.