STRUCTURE/INTERACTIONS OF ACTINS & ACTIN-BINDING PROTEIN

这个项目的长期目标是确定在原子 精选蛋白质的三维结构解析 肌动蛋白系统，期望这些信息将 提供了了解肌动蛋白为基础的收缩和 细胞骨架系统的组装及其功能。至 为了实现这一目标，我们提出了下一步的目标如下 五年了。收集更多的X射线数据并改进结构 将棘阿米巴Profilin-I的分辨率提高到2.0A或更高。 分析可能的结构和顺序上的相似性 Profilin和其他肌动蛋白结合蛋白以及Profilin和 溶菌酶，使用比较DNA或蛋白质的程序 序列或用于匹配结构。确定结构 现有的棘阿米巴Profilin-II晶体的分辨率为 2.4A或更好，使用自然x射线衍射数据和分子 替换为计算初始阶段。确定绑定 多聚脯氨酸的X射线结晶学研究：Profilin-I上的S位点 共晶体。人血小板膜蛋白结构的测定 以原子分辨率通过分子置换或重原子 原子方法。当此信息可用时，将用于 协助测定脊椎动物肌动蛋白的结构- Profilin Compelx。人血小板膜蛋白的小晶体 已经有货了。棘阿米巴的结构测定 Actophorin，一种15,000道尔顿肌动蛋白细丝的切断蛋白，由x- 多重原子同象的射线结晶学 替换以计算初始阶段。小晶体 Actophorin已经上市了。继续我们令人鼓舞的最初阶段 制备适用于x射线衍射的其他材料的晶体的努力 细胞骨架中的蛋白质包括棘阿米巴肌动蛋白， 棘阿米巴与肌动蛋白的共价交联络合物 Profilin-I或Profilin-II，以及7万个道尔顿头骨碎片 棘阿米巴肌球蛋白-II。我们还将尝试使 兔心肌肌球蛋白亚段-1及其必需和 肌球蛋白的调节轻链。我们将尝试 使卵子蛋白在肌动蛋白系统中结晶 可用。利用电子进行三维重建 不同方法制备的肌动蛋白单丝的显微照片 努力达成细丝的共识模型，并 确定聚合条件是否影响 以某种微妙的方式显示聚合物的结构。提供 棘阿米巴轮廓蛋白和详细的结构信息 将使用二维核磁共振研究的合作者 分子的溶液结构及其对结合的响应 多聚脯氨酸和肌动蛋白多肽与我们的X射线进行比较 晶体结构。

The long range goal of this project is to determine at atomic resolution the three-dimensional structures of selected proteins of the actin system with the expectation that this information will provide the key to understanding how actin-based contractile and cytoskeletal systems are assembled ad how they function. To reach this goal we propose the following objectives for the next five years. Collect additional x-ray data and refine the structure of Acanthamoeba profilin-I to a resolution of 2.0 A or better. Analyze possible structural and sequential similarities between profilin and other actin-binding proteins and between profilin and lysozyme, using programs for comparing DNA or protein sequences or for matching up structures. Determine the structure of existing crystals of Acanthamoeba profilin-II at a resolution of 2.4A or better using native x-ray diffraction data and molecular replacement ot calculate initial phases. Determine the binding sites(s) on profilin-I for polyproline by x-ray crystallography of co-crystals. Determine the structure of human platelet profilin at atomic resolution by either molecular replacement or heavy atom methods. When available this information will be used to assist with determination of the structure of the vertebrate actin- profilin compelx. Small crystals of human platelet profilin are already available. Determine the structure of Acanthamoeba actophorin, a 15,000 Dalton actin filament severing protein, by x- ray crystallography using multiple heavy atom isomorphous replacements to calculate initial phases. Small crystals of actophorin are already available. Continue our encouraging initial efforts to prepare crystals, suitable for x-ray diffraction, of other proteins in the cytoskeleton including Acanthamoeba actin, covalently crosslinked complexes of Acanthamoeba actin with profilin-I or profilin-II, and a 70,000 Dalton fragment of the head of Acanthamoeba myosin-II. We will also attempt to crystalize rabbit cardiac muscle myosin subfragment-1 and the essential and regulatory light chains of smooth muscle myosin. We will attempt to crystalize oather proteins in the actin system as they become available. Make 3-dimensional reconstructions from electron micrographs of single actin filaments prepared in various ways in a effort to arrive at a consensus model for the filaments and to determine whether the polymerization conditions influence the structure of the polymer in some subtle way. Provide Acanthamoeba profilin and detailed structural information to collaborators who will use two-dimensional NMR to investigate the solution structure of the molecule and its repsonse to binding of polyproline and actin peptides for comparison with our x-ray crystallographic structure.