TR4 ANALYSIS IN THE PRESENCE AND ABSENCE OF CALCIUM

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 BAD-1是双相真菌皮炎芽生菌的致病决定因子。 它装饰酵母细胞的表面，可以从培养上清液中分离出来。 其中BAD-1基因已被敲除的酵母是减毒的且非致病性的。 BAD 1介导致病性的机制还不完全清楚，尽管其序列含有30-35个串联排列的EF-手状结构域。 BAD-1已被证明与这些推定的EF-手结构域中的每一个结合大约一个钙离子（总共约30个）。 我们提出了一个推定的BAD 1钙结合域，将在结构上类似于哺乳动物血小板反应蛋白（TSP）的钙结合域。TSP EF-手状域缺乏稳定？-螺旋，而是依靠半胱氨酸键来固定钙结合环。BAD-1也缺少一个？- 螺旋稳定基序，但确实包括两个半胱氨酸残基，其包含每个串联重复序列。 BAD-1中提出的环结构可能被限制在SYBYL中的血小板反应蛋白环的结构中，并且确定键能最小化计算在该模型中是非常有利的。 然而，这种结构从未得到证实，BAD-1蛋白质抵抗了结晶的尝试。 由于BAD-1本身是一个120 kDa的大蛋白，我们已经设计了一小部分的串联重复序列，包括3.5个重复的推定的钙结合结构域。 这种蛋白质，称为TR 4，已经从E. coli表达系统中表达，并且在纯化后以半胱氨酸交联的单一构象迁移到天然PAGE凝胶上。 我们希望双标记TR 4，将其重新折叠成天然构象，然后通过NMR光谱分析这种结构。 初步的N15光谱表明，我们已经成功地在TR 4中重建了三个结构相似的串联重复序列，并且该蛋白质在高浓度下足够稳定，可用于扩展分析

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