STRUCTURAL STUDIES OF PHOSPHATASES, AND PARVALBUMINS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 本项目研究了两种含有碱土金属结合部位的金属蛋白：C类酸性磷酸酶和小白蛋白。C类酸性磷酸酶是一类外膜酸性磷酸酶，它含有四个必需的天冬氨酸残基，位于多肽链N-末端的[IV]-[Val]-D-[IL]-D-E-T-[Vm]-L-X-[NT]-X(2)-Y和C-末端的[IV]-[LM]-X(2)-G-D-[NT]-L-X-D-F序列中。活性中心的特点是镁离子与三个保守的Asp残基结合。由于CCAP定位于细菌外膜，是疫苗开发的潜在候选者，利用流感嗜血杆菌CCAP的催化失活突变体创建针对非分型流感嗜血杆菌的疫苗已取得一些进展。我们正在研究包括流感嗜血杆菌、图拉氏杆菌和炭疽杆菌在内的病原菌的CCAP的结构、功能和免疫原性，以了解这些酶在毒力中的作用，并为设计用于重组疫苗的免疫原性增强的失活突变体提供平台。该项目的第二个方面专注于小蛋白，这是一种小的(MR=12,000)脊椎动物特有的EF-Hand蛋白。小白蛋白家族包括α和β亚类，通过等电点和谱系特异性序列差异来区分。尽管它们的金属-离子结合位置和整体折叠总体上相似，但小白蛋白在二价离子亲和力上表现出广泛的差异。我们正在使用特定的小白蛋白亚型来探索这些差异的物理和结构基础。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Two metalloproteins containing alkaline earth metal binding sites were investigated in this project: class C acid phosphatases and parvalbumins. Class C acid phosphatases (CCAPs) are outer membrane acid phosphatases that contain four essential Asp residues imbedded in the bipartite sequence motif of [IV]-[VAL]-D-[IL]-D-E-T-[VM]-L-X-[NT]-X(2)-Y in the N-terminal half of the polypeptide chain and [IV]-[LM]-X(2)-G-D-[NT]-L-X-D-F in the C-terminal half. The active site features a magnesium ion bound to three of the conserved Asp residues. Because of their localization to the bacterial outer membrane, CCAPs are potential candidates for vaccine development, and some progress has been made toward creating a vaccine against nontypeable Haemophilus influenzae using catalytically inactive mutants of the H. influenzae CCAP. We are studying the structure, function, and immunogenicity of CCAPs from pathogenic bacteria including H. influenzae, F. tularensis, and B. anthracis in order to understand the role of these enzymes in virulence and to provide a platform for the design of inactive mutants with enhanced immunogenicity for use in recombinant vaccines. The second aspect of this project focuses on parvalbumins, which are small (Mr=12,000), vertebrate-specific EF-hand proteins. The parvalbumin family includes alpha and beta sublineages, distinguished by isoelectric point and lineage-specific sequence differences. Despite the general similarity of their metal-ion binding sites and overall fold, parvalbumins exhibit broad variations in divalent ion affinity. We are exploring the physical and structural basis for these differences, using specific parvalbumin isoforms.