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， 期望这些信息将 提供了理解肌动蛋白为基础的收缩和 细胞骨架系统的组装以及它们的功能。 到 为实现这一目标，我们提出了以下目标 五年 收集额外的X射线数据并优化结构 棘阿米巴profilin-I的分辨率为2.0 A或更好。 分析可能的结构和顺序相似之处， profilin和其它肌动蛋白结合蛋白之间以及profilin和 溶菌酶，使用程序比较DNA或蛋白质 序列或用于匹配结构。 确定结构 的现有晶体的阿米巴profilin-II的分辨率 2.4A或更好，使用天然X射线衍射数据和分子 替换或计算初始相位。 确定结合 profilin-I上的多聚脯氨酸位点（通过X射线晶体学） 共晶。 人血小板前纤维蛋白的结构测定 在原子分辨率的分子置换或重 原子方法 这些信息将用于 帮助确定脊椎动物肌动蛋白的结构， 复合型profilin 人血小板前纤维蛋白的小晶体 已经有了。 确定阿米巴的结构 肌动蛋白，一种15，000道尔顿的肌动蛋白丝切割蛋白，通过x- 多重重原子同晶X射线晶体学 替换计算初始相位。 的小晶体 actophorin已经存在。 继续我们令人鼓舞的初步 努力制备适合于X射线衍射的其他晶体 细胞骨架中的蛋白质包括阿米巴肌动蛋白， 阿米巴肌动蛋白的共价交联复合物 profilin-I或profilin-II，以及头部的70，000道尔顿片段 阿米巴肌球蛋白-II。 我们还将尝试 兔心肌肌球蛋白亚片段-1和必需的， 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质结晶， available. 从电子进行三维重建 用各种方法制备的单个肌动蛋白丝的显微照片， 努力达成一个共识模型的细丝， 确定聚合条件是否影响 聚合物的结构。 提供 阿米巴profilin和详细的结构信息， 合作者将使用二维核磁共振来研究 分子的溶液结构及其对结合的反应 与我们的X光片进行对比 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.