TR4 ANALYSIS IN THE PRESENCE AND ABSENCE OF CALCIUM

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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. BAD-1 is a pathogenic determinant for the dimorphic fungus Blastomyces dermatitidis. It decorates the surface of yeast cells and may be isolated from culture supernates. Yeast in which the gene for BAD-1 has been knocked out are attenuated and non-pathogenic. The mechanisms by which BAD1 mediates pathogenicity are not full understood, although its sequence contains 30-35 EF-hand-like domains arrayed in tandem. BAD-1 has been shown to bind approximately one calcium ion for each of these putative EF-hand domains (~30 in all). We propose a putative BAD1 calcium-binding domain that would be structurally similar to the calcium-binding domain of mammalian thrombospondin (TSP). TSP EF-hand-like domains lack a stabilizing ?-helix and rely instead on cysteine bonds to secure the calcium-binding loop. BAD-1 also lacks an ? -helix stabilizing motif, but does include two cysteine residues which bracket each tandem repeat. The proposed loop structure in BAD-1 may be constrained to the structure of the thrombospondin loop in SYBYL, and bond energy minimization calculations were determined to be very favorable in this model. This structure has never been verified, however, and the BAD-1 protein has resisted attempts at crystallization. As BAD-1 itself is a large protein of 120 kDa, we have engineered a small portion of the tandem repeat sequence that includes 3.5 repeats of the putative calcium-binding domain. This protein, termed TR4, has been isolated and refolded from an E. coli expression system, and upon purification migrates on a native PAGE gel in a single conformation of cysteine cross-linking. We hope to double-label TR4, refold it into its native conformation, and then analyze this structure via NMR spectroscopy. Preliminary N15 spectra demonstrate that we have succeeded in recreating three structurally similar tandem repeats in TR4 and that the protein is sufficiently stable at high concentrations for extended analysis