STRUCTURAL BIOLOGY OF MACROMOLECULAR COMPLEXES

许多重要的细胞功能是由大分子执行的 复合体。这个项目的目标是阐明这些结构， 这种络合物的组装性质和相互作用，重点是 它们的功能内涵。 (1)噬菌体尾部纤维是一类新兴的低聚物 富含b-折叠的具有新构象的纤维蛋白， 糖类结合特性，以及模块化蛋白的明显应用范围 工程学。我们已经开发出了我们之前报道的三b-螺旋 通过定义五种T-偶数纤维的主域结构建立T-偶数纤维模型 用计算机增强电子显微镜观察噬菌体。各个域都有 在大肠杆菌中表达，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)角化细胞包膜(CEs)，由共价的 交联化蛋白，增强保护性和不透性 表皮和其他层状鳞状组织中角质细胞的特性 上皮细胞。我们正在用几个电子显微镜研究CE结构 (EM)方法，并为其组装开发了一种新的模式。硫磺 通过电子光谱成像获得的图谱确定了氯氰菊酯(a 富含半胱氨酸的蛋白质)在细胞质颗粒和CE中，证实 更早的免疫细胞化学数据。他们还估计了氯氰菊酯的含量 约75%，与以下给出的数字一致 分离的CES氨基酸组成的数学模型。 单位面积质量的扫描透射法测量 用常规透射法测得的厚度值非常均匀 分别为7 kDa/nm2和14.5 nm。为了解释这些问题，我们 假设CE的外层只有一种氯氰菊酯 分子厚，以及其他蛋白质之间的氯丙烷交联物 是CE生物力学特性的重要决定因素。 (3)依赖能量的胞内蛋白分解的主要部分是 由CLP的蛋白水解酶家族执行，通常包括 蛋白水解组分和三磷酸腺苷水解组分。后者 蛋白质被认为能够识别底物，将其展开，并为其提供食物 进入蛋白水解酶，它是中空壳，其内部容纳着 活动站点。我们正在研究这些物质的结构特性 特别注意旋转对称性的分子和 参与形成活性络合物的相互作用。在过去 年，我们发现ClpY ATPase和ClpQ蛋白酶都是 六角体，以便它们的关联不会引发对称失配 存在于7倍的ClpP蛋白酶和6倍的ClpA ATPase之间。一个 ClpA的三维密度图清楚地显示了它的两个六角体 结构域的层级；ClpA与REPA蛋白络合的图像 表明该二聚体底物结合在ClpA中心附近 六角星。

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.