Characterization of the flexible portions of fibrinogen

纤维蛋白原柔性部分的表征

• 批准号: 6962377
• 负责人: RUSSELL F DOOLITTLE
• 金额: $ 19.25万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-05 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6962377
• 关键词: Agnatha; SDS polyacrylamide gel electrophoresis; animal tissue; biochemistry; chickens; clinical research; crosslink; fibrin; fibrin stabilizing factor; fibrinogen; fibrinopeptide; human subject; molecular biology; protein purification; protein structure function; structural biology; thrombin

DESCRIPTION (provided by applicant): Although recently reported three-dimensional structures of fibrinogen and its core fragments have been very revealing, key regions of these molecules are inherently flexible and have not yielded to the X-ray approach. These mobile regions, which amount to almost one-third of the molecule, are extremely important in a functional sense, and ignorance of their whereabouts is a distinct limitation to understanding fibrin clot formation. The mobile regions include the amino-terminal segments of the alpha and beta chains where the thrombin-sensitive bonds occur, the carboxyl-terminal segment of the alpha chain where factor XIII-catalyzed gamma-gamma dimer formation takes place, and, finally, the entire carboxyl-terminal section of the alpha chain, commonly called the alphaC domain. The latter plays a poorly understood role in the association of protofibrils and also involves factor XIII-induced cross-links. The goal of the current project is to establish the whereabouts of the flexible regions in native fibrinogen by using conventional but carefully crafted biochemistry experiments. The experiments fall into two general realms, both employing the same general procedures. In one set of experiments the biochemistry is conducted directly on crystals of native fibrinogen (chicken) where the bulk of the molecule is constrained and only the regions of interest are able to move about and interact. Thrombin and other specific enzymes will be used to specifically remove the fibrinopeptides directly in the crystal, after which selective chemical cross-linking will be used to join the newly exposed A and/or B knobs within each molecule. Similar experiments will be conducted on fibrin (human), where the constraints are somewhat different. These studies are aimed at finding how far apart the mobile knobs are from each other, on the average. Other experiments will be performed that immobilize the a chain carboxyl regions in situ, again both in crystals of native fibrinogen and in fibrin clots, by taking advantage of easily reduced disulfide bonds. The goal here is to find how the alphaC domains are situated relative to each other. In a related study, hybrid fibrins prepared from fibrinogens from different species will be used to follow polymerization events, especially the interactions and cross-linking of gamma-chain carboxyl terminal segments on the one hand and alphaC domains on the other. These studies are aimed at determining the degree of specificity involved in the assembly of fibrin units and protofibrils as well as the relative locations of the regions involved.

描述（由申请人提供）：尽管最近报道的纤维蛋白原及其核心片段的三维结构非常具有启示性，但这些分子的关键区域固有地具有柔性，并且不屈服于X射线方法。这些移动的区域几乎占分子的三分之一，在功能意义上是极其重要的，而对其位置的无知是理解纤维蛋白凝块形成的明显限制。移动的区域包括α和β链的氨基末端片段，其中发生凝血酶敏感性键，α链的羧基末端片段，其中发生因子XIII催化的γ-γ二聚体形成，最后是α链的整个羧基末端部分，通常称为α C结构域。后者起着知之甚少的作用，在协会的原纤维，也涉及因子XIII诱导的交联。目前项目的目标是通过使用常规但精心制作的生物化学实验来确定天然纤维蛋白原中柔性区域的位置。实验分为两个一般领域，都采用相同的一般程序。在一组实验中，生物化学直接在天然纤维蛋白原（鸡）的晶体上进行，其中大部分分子受到限制，只有感兴趣的区域能够移动和相互作用。凝血酶和其他特异性酶将用于直接特异性地去除晶体中的纤维蛋白肽，之后将使用选择性化学交联来连接每个分子内新暴露的A和/或B节。将在纤维蛋白（人）上进行类似的实验，其中约束条件略有不同。这些研究的目的是找出移动的旋钮之间的平均距离。还将进行其他实验，利用容易还原的二硫键，在天然纤维蛋白原晶体和纤维蛋白凝块中原位固定α链羧基区域。这里的目标是找到alphaC结构域如何相对于彼此定位。在相关研究中，从来自不同物种的纤维蛋白原制备的杂合纤维蛋白酶将用于跟踪聚合事件，特别是一方面γ链羧基末端片段和另一方面alphaC结构域的相互作用和交联。这些研究旨在确定纤维蛋白单位和原纤维组装的特异性程度以及所涉及区域的相对位置。