FIBRINOGEN CRYSTALLIZATION REQUIRES LIMITED PROTEOLYSIS

对血液凝固及其功能障碍的全面了解， 心血管疾病需要详细的结构信息， 和纤维蛋白原分子的相互作用。 一个主要目标是 实验室是确定的三维结构的 纤维蛋白原的X射线晶体学。 尽管付出了巨大的努力， 纤维蛋白原尚未结晶。 牛的有限消化 纤维蛋白原与来自铜绿假单胞菌的粗蛋白酶提取物， 然而，这导致产生的晶体的折射率为约6。 本提案的主要目的是确定 形成这些的蛋白酶修饰的纤维蛋白原的一级结构 晶体 为了执行这项建议，必须 对假单胞菌蛋白酶进行分离和鉴定。 表征 这种新的酶将包括确定其氨基酸组成， 切割特异性和对酶抑制剂的敏感性。 的 用纯化和表征的纤维蛋白原修饰纤维蛋白原的优点 Ps-1的制备包括更好地控制消化条件， 以及降低裂解异质性。 一次消化条件 已经建立了各种溶剂系统，离子强度， 温度和pH值范围将进行探索，以找到组合， 一致地导致良好有序晶体的良好供应。 尝试 也将用来生产高度有序的人类纤维蛋白原晶体 用假单胞菌蛋白酶修饰后。 这其中的主要焦点 建议将是对一级结构变化的决定 纤维蛋白原分子形成晶体所需的量。 四级 方法将被使用：1）分离片段和肽释放由 用Ps-1有限消化，2）氨基酸分析，3）氨基末端 序列测定，和4）将多肽放置在 纤维蛋白原的已知结构。 修饰的一级结构 晶体中的纤维蛋白原将从这些数据的分析中推导出来。 总之，这些研究对于获取和解释 修饰纤维蛋白原晶体的电子密度图。 知识 需要纤维蛋白原的详细结构来建立关键的 决定这些分子在纤维蛋白中堆积的相互作用 凝块

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.