STRUCTURE/INTERACTIONS OF ACTINS & ACTIN-BINDING PROTEIN

该项目的长期目标是确定原子 解析所选蛋白质的三维结构 肌动蛋白系统，期望该信息将 提供理解基于肌动蛋白的收缩和 细胞骨架系统的组装及其功能。 至 为实现这一目标，我们提出以下目标： 五年。 收集额外的 X 射线数据并完善结构 棘阿米巴 profilin-I 的分辨率达到 2.0 A 或更高。 分析之间可能的结构和顺序相似性 Profilin 和其他肌动蛋白结合蛋白以及 Profilin 和其他肌动蛋白结合蛋白之间 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 现有棘阿米巴 profilin-II 晶体的分辨率为 2.4A 或更好，使用原生 X 射线衍射数据和分子 替换 ot 计算初始阶段。 确定绑定 通过 X 射线晶体学分析聚脯氨酸的 profilin-I 上的位点 共晶。 确定人血小板谱蛋白的结构 通过分子置换或重原子分辨率 原子方法。 当可用时，此信息将用于 协助确定脊椎动物肌动蛋白的结构 轮廓蛋白复合物。 人血小板分布蛋白的小晶体是 已经可用。 确定棘阿米巴的结构 Actophorin，一种 15,000 道尔顿的肌动蛋白丝切断蛋白，由 x- 使用多个重原子同晶的射线晶体学 替换来计算初始阶段。 小晶体的 肌动蛋白已经可用。 继续我们令人鼓舞的初始 努力制备适用于 X 射线衍射的其他晶体 细胞骨架中的蛋白质，包括棘阿米巴肌动蛋白， 棘阿米巴肌动蛋白与的共价交联复合物 profilin-I 或 profilin-II，以及 70,000 道尔顿的头部片段 棘阿米巴肌球蛋白-II。 我们也会尝试去结晶 兔心肌肌球蛋白亚片段-1和必需和 平滑肌肌球蛋白的调节轻链。 我们将尝试 使肌动蛋白系统中的其他蛋白结晶 可用。 从电子进行 3 维重建 以各种方式制备的单肌动蛋白丝的显微照片 努力达成细丝的共识模型并 确定聚合条件是否影响 聚合物的结构以某种微妙的方式。 提供 棘阿米巴谱和详细的结构信息 将使用二维核磁共振进行研究的合作者 分子的溶液结构及其对结合的反应 聚脯氨酸和肌动蛋白肽与我们的 X 射线比较 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.