FIBRINOGEN CRYSTALLIZATION REQUIRES LIMITED PROTEOLYSIS

对某些疾病的凝血及其功能障碍的全面认识 心血管疾病需要关于结构的详细信息 以及纤维蛋白原分子之间的相互作用。这样做的一个主要目标是 实验室是对三维结构的测定 纤维蛋白原的X射线结晶学分析。尽管付出了广泛的努力，但本地化 纤维蛋白原尚未结晶。牛的有限消化 纤维蛋白原和来自铜绿假单胞菌的粗酶提取物， 然而，结果是产生了衍射率约为6埃的晶体。 这项提案的主要目标是确定在 形成这些的蛋白酶修饰的纤维蛋白原的一级结构 水晶。为了执行这项提议，有必要 分离和鉴定假单胞菌蛋白水解酶。特征描述 这种新型的酶将包括其氨基酸组成的测定， 切割特异性和对酶抑制剂的敏感性。这个 一种纯化和表征的纤维蛋白原修饰的优点 PS-1的制备包括更好地控制消化条件，因为 降低解理的非均质性。一次消化条件 已经建立，各种溶剂系统，离子强度，和 我们将探索温度和pH范围，以找到 始终如一地产生良好的有序晶体供应。尝试 也将产生高度有序的人类纤维蛋白原晶体 用假单胞菌蛋白水解酶修饰后。这件事的主要焦点是 提案将确定初级结构的变化 分子形成晶体所需的纤维蛋白原。四个阶段 将使用的方法：1)分离由 PS-1限制性消化，2)氨基酸分析，3)氨基端 序列确定，以及4)将多肽放置在 纤维蛋白原的已知结构。改性体的一级结构 对这些数据的分析将推断出晶体中的纤维蛋白原。 综上所述，这些研究对于获得和解释是必不可少的 修饰的纤维蛋白原晶体的电子密度图。了解以下内容 需要纤维蛋白原的详细结构来确定关键的 决定这些分子在纤维蛋白中堆积的相互作用 血块。

A complete understanding of blood clotting and its malfunction in certain cardiovascular diseases requires detailed information about the structure and interactions of the fibrinogen molecule. A major goal of this laboratory is the determination of the three-dimensional structure of fibrinogen by X-ray crystallography. Despite extensive efforts, native fibrinogen has not yet been crystallized. Limited digestion of bovine fibrinogen with a crude protease extract from Pseudomonas aeruginosa, however, results in the production of crystals that diffract to about 6 A. The major objective of this proposal is to determine the modifications in the primary structure of the protease-modified fibrinogen that forms these crystals. In order to carry out this proposal, it will be necessary to isolate and characterize the Pseudomonas protease. Characterization of this novel enzyme will include determination of its amino acid composition, cleavage specificity, and susceptibility to enzymatic inhibitors. The advantages of modifying fibrinogen with a purified and characterized preparation of Ps-1 include greater control of digestion conditions, as well as reduction of cleavage heterogeneity. Once digestion conditions have been established, various solvent systems, ionic strengths, and temperature and pH ranges will be explored to find the combination which consistently results in a good supply of well-ordered crystals. Attempts will also be made to produce highly-ordered crystals of human fibrinogen after modification with the Pseudomonas protease. The major focus of this proposal will be the determination of the changes in the primary structure of fibrinogen required for the molecule to form crystals. A four-stage approach will be used: 1) isolation of fragments and peptides released by limited digestion with Ps-1, 2) amino acid analysis, 3) amino-terminal sequence determination, and 4) placement of the polypeptides within the known structure of fibrinogen. The primary structure of the modified fibrinogen in crystals will be deduced from an analysis of these data. Taken together, these studies are essential for obtaining and interpreting electron density maps of the modified fibrinogen crystals. Knowledge of the detailed structure of fibrinogen is required to establish the critical interactions that determine the packing of these molecules in the fibrin clot.