STRUCTURAL BIOLOGY OF MACROMOLECULAR COMPLEXES

大分子复合物的结构生物学

• 批准号: 6160813
• 负责人: A C STEVEN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6160813
• 关键词: active sites; adenosinetriphosphatase; bioenergetics; biomechanics; conformation; crosslink; endopeptidases; enzyme substrate complex; fibrous protein; image enhancement; keratinocyte; membrane proteins; protein folding; protein structure function; scanning transmission electron microscopy; structural biology; transmission electron microscopy; virus protein

Many important cellular functions are performed by macromolecular complexes. The goal of this project is to elucidate the structures, assembly properties, and interactions of such complexes with emphasis on their functional connotations. (1) Bacteriophage tail-fibers are emerging as a class of oligomeric fibrous proteins with novel conformations rich in b-sheets, saccharide-binding properties, and evident scope for modular protein engineering. We have developed our previously reported triple-b-helix model of T-even fibers by defining the domainal structures of five such phages by computer-enhanced electron microscopy. Individual domains have been expressed in E. coli and induced to trimerize and fold correctly by fusing them with a trimer-forming domain from another protein. (2) Cornified cell envelopes (CEs), consisting of sheets of covalently cross-linked protein, enhance the protective and impenetrability properties of corneocytes in the epidermis and other stratified squamous epithelia. We are studying CE structure by several electron microscopic (EM) approaches and have developed a new model for their assembly. Sulfur maps obtained by electron spectroscopic imaging identify loricrin (a cysteine-rich protein) in cytoplasmic granules and in the CE, confirming earlier immunocytochemical data. They also estimate the loricrin content of CEs in situ as about 75%, consistent with the figure given by mathematical modeling of amino acid compositions of isolated CEs. Measurements of mass-per-unit-area by scanning transmission EM and of thickness by conventional transmission EM gave remarkably uniform values of 7 kDa/nm2 and 14.5 nm, respectively. To account for them, we hypothesize that the outer portion of the CE is only one loricrin molecule thick, and that inter-loricrin cross-linking by other proteins is an important determinant of the CE's biomechanical properties. (3) A major portion of energy-dependent intracellular proteolysis is carried out by the Clp family of proteases, which generically consist of a proteolytic component and an ATP-hydrolyzing component. The latter proteins are thought to recognize substrates, unfold them, and feed them into the proteases which are hollow shells, whose interiors house the active sites. We are studying the structural properties of these molecules with particular attention to rotational symmetry and the interactions involved in the formation of active complexes. In the past year we have found that both the ClpY ATPase and the ClpQ protease are hexamers, so that their association does not invoke the symmetry mismatch that exists between 7-fold ClpP protease and 6-fold ClpA ATPase. A 3-dimensional density map of ClpA clearly visualizes its two hexameric tiers of domains; and images of ClpA complexed with RepA protein indicates that this dimeric substrate binds near the center of the ClpA hexamer.

许多重要的细胞功能是由大分子 配合物这个项目的目标是阐明结构， 组装性能，以及这种复合物的相互作用，重点是 其功能内涵。 (1)噬菌体尾纤维作为一类寡聚体 具有富含b-折叠的新构象的纤维蛋白， 糖结合特性和模块蛋白的明显范围 工程.我们已经开发了我们以前报道的三b螺旋 T偶纤维模型，通过定义五个这样的畴结构， 通过计算机增强电子显微镜观察。各个域具有 在E.大肠杆菌，并诱导三聚体和正确折叠， 将它们与来自另一种蛋白质的三聚体形成结构域融合。 (2)皮质细胞包膜（CE），由共价连接的 交联蛋白，增强保护性和不渗透性 表皮和其他复层鳞状上皮中角质细胞的性质 上皮细胞我们正在用几种电子显微镜研究CE结构 (EM)方法，并开发了一个新的模型，其组装。硫 通过电子光谱成像获得的图谱鉴定了兜甲蛋白（a 富含半胱氨酸的蛋白质），证实 早期的免疫细胞化学数据。他们还估计了兜甲蛋白的含量 的CE在原位约为75%，与由 分离的CE的氨基酸组成的数学建模。 扫描透射电磁法测量单位面积质量 厚度由传统的透射电磁给出了非常均匀的值 7 kDa/nm ~ 2和14.5nm。为了解释它们，我们 假设CE的外部部分仅为一个兜甲蛋白 分子厚，且甲膜蛋白间通过其它蛋白质交联 是CE生物力学性能的重要决定因素。 (3)能量依赖性细胞内蛋白水解的主要部分是 由蛋白酶的Clp家族进行，其一般由以下组成： 蛋白水解组分和ATP水解组分。后者 蛋白质被认为能够识别底物，展开底物，并为底物提供营养 蛋白酶是中空的外壳，其内部容纳 活性位点。我们正在研究这些 分子，特别注意旋转对称性， 参与形成活性复合物的相互作用。过去 一年来，我们发现ClpY ATP酶和ClpQ蛋白酶都是 六聚体，因此它们的结合不会引起对称性错配， 存在于7倍ClpP蛋白酶和6倍ClpA ATP酶之间。 一 3-ClpA的三维密度图清楚地显示了它的两个六聚体 结构域的层次;以及与RepA蛋白复合的ClpA的图像 表明这种二聚底物结合在ClpA的中心附近 六聚体