STRUCTURES OF CATALYTIC DOMAINS OF BLOOD PROTEINS

这项工作的直接目标是解决和完善结构 人PPACK-α-凝血酶的合成 这种丝氨酸原型的结构 蛋白酶催化结构域将揭示的方式，其中高度 实现了选择性的纤维蛋白原-凝血酶相互作用。 此外，委员会还认为， 由于血液凝固/纤维蛋白溶解的许多酶域 高度同源的人α-凝血酶结构将用于 模拟这些其他蛋白酶的密切同源区域， 物种之间的差异。 然后，α-凝血酶的结构将用于分子生物学。 置换应用，以解决水蛭素复合物的结构 和PACK-alpha-凝血酶。 这些研究旨在 定位抗血栓水蛭素和 凝血酶分子描绘三维和其他因素 这就是为什么这些蛋白质之间 分子（分别为毫微微和皮科摩尔）。 类似的方法将 用于确定凝血酶原1和单链的结构 凝血酶原2。 在前一种情况下，旋转平移搜索 也将包括三个二硫化物，三环的Kringle 2- 凝血酶原的催化结构域相互作用; 凝血酶2和凝血酶结构将揭示构象变化 在凝血因子Xa活化凝血酶原裂解时发生。 这项工作的一个较长范围的推力将集中在结构上 确定完整的盒结构域分子，例如 血栓溶解剂组织纤溶酶原激活物（TPA）和尿激酶原。 的 努力的范围将得到扩大和加强， 第三代结晶，去糖基化外显子重排 TPA分子的构建体。 的可能性的数量 交换和/或删除指、生长因子、Kringle和 催化域是有吸引力的大，因此有利地增加了 发现可结晶结构的概率。

The immediate objective of this work is to solve and refine the structure of human PPACK-alpha-thrombin. The structure of this prototype of serine protease catalytic domains will reveal the manner in which the highly selective fibrinogen-thrombin interaction is accomplished. Moreover, since many of the enzyme domains of blood coagulation/fibrinolysis are highly homologous, the human alpha-thrombin structure will be used to model closely homologous regions of these other proteases as well as model differences between species. The structure of alpha-thrombin will then be used in molecular replacement application to solve the structures of the hirudin complexes of alpha- and PPACK-alpha-thrombin. These studies are designed to localize the binding sites of the anti-thrombotic hirudin and the thrombin molecules to delineate the three dimensional and other factors responsible for the incredibly singular binding affinity between these molecules (femto and pico molar, respectively). Similar methods will be employed to determine the structures of prothrombin 1 and single chain prothrombin 2. In the case of the former, rotation-translation searches will also include the three disulfide, triple loop of the kringle 2- catalytic domain interactions of prothrombin; a comparison of the pre- thrombin 2 and thrombin structures will reveal the conformational changes occurring upon activation cleavage of prothrombin by Factor Xa. A longer range thrust of the work will center upon the structure determination of intact cassette domain molecules such as the thrombolytics tissue plasminogen activator (TPA) and prourokinase. The scope of the effort will be broadened and enhanced by also attempting the crystallization of third generation, deglycosylated exon shuffled constructs of the TPA molecule. The number of possibilities of interchanging and/or deleting finger, growth factor, kringle and catalytic domains is attractively large thus favorably increasing the probability of uncovering crystallizable constructs.