Purification and Crystallization of a Putative PhoH-like Protein from C. diphtheriae. TIFFANY HOAGE (University of Wisconsin-Stout Menomonie, WI 54751) ANDRZEJ JOACHIMIAK (Argonne National Laboratory, Argonne, IL, 60439)

One of the goals of the Midwest Center for Structural Genomics (MCSG) is to determine the structure of novel and important eukaryotic and bacterial proteins having specific amino-acid sequences and unknown functions. By characterizing the structures, functions of the proteins may be better understood, new protein folds may be identified, and in some cases new pharmaceuticals can be developed. One of the proteins currently under study at the MCSG in collaboration with the Structural Biology Center (SBC) at Argonne National Laboratory is a putative PhoH-like protein naturally found in Corynebacterium diphtheriae, MSCG code APC 82804. It represents a unique class of phosphate starvation-inducible proteins with putative ATPase activity. To determine the protein's structure using x-ray crystallography, crystals that diffract x-rays to at least 3 angstroms (Å) resolution are required. A crystallization condition that produces macroscopic crystals has now been found for APC 82804. The protein used for crystallization was expressed from the recombinant plasmid pMCSG7 that contained the gene coding for APC 82804 in Escherichia coli host strains BL21-Gold (DE3). The protein was purified on a Ni-NTA column. Mosquito protein crystallization robot was used to screen for crystallization conditions. Crystals formed in wells containing 2.0 M sodium formate and buffer at pH 7.0 and 8.5. These conditions were further optimized, one of which produced macroscopic crystals that diffracted x-rays to 5.25 Å resolution. Further optimization will be necessary to improve crystallization for structure determination. Once the structure is obtained, it can then be analyzed to determine the function of APC 82804 within C. diphtheriae and possibly be used to prevent the bacteria's toxic effects in humans.