NMR STUDIES ON PLASMINOGEN: STRUCTURE AND DYNAMICS

纤溶酶原的核磁共振研究：结构和动力学

• 批准号: 3340526
• 负责人: MIGUEL LLINAS
• 金额: $ 17.61万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-07-01 至 1990-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3340526
• 关键词: antifibrinolytic agents; chemical association; chemical binding; chemical structure function; conformation; fibrin; human tissue; intermolecular interaction; lysine; molecular rearrangement; nuclear magnetic resonance spectroscopy; plasminogen; plasminogen activator; thermodynamics

It is proposed to continue research on the conformational and dynamic properties of plasminogen (92,000 Mr), with emphasis in understanding the structrure, function and biochemical regulation of the plasminogen heavy chain, in particular of its five kringles. Plasminogen kringles are 10,000 Mr strutural and folding domains constrained by three Cys-Cys bridges; they are responsible for anchoring plasminogen to fibrin and to blood clots, and probably mediate interactions with alpha 2-antiplasmin and with the plasminogen pre-activation peptide (haemostasis control factors). These interactions are thought to involve a lysine-binding site (LBS), known to be carried to kringle 1 (K1), K2 and, in all likelihood, by K5. K1, K4, K5 and the combined K2+3 are isolated by proteolytic cleavage of plasminogen extracted from human blood plasma. The kringles are then subjected to 1-D and 2-D NMR spectroscopy at 300 and 600 MHz (1H frequencies) both in 1H2O and in 2H2O. Particular emphasis will be placed on systematically characterizing the structural/steric requirements of ligand drugs and of short model synthetic polypeptide chains containing C-terminal L-Lys. From Overhauser studies and other chemical evidence on K4, it is proposed that the LBS is structured by the Cys51 -Cys75 loop, and involves polar residues Asp57 and Arg71, and the aromatic side chains of Phe 64 and Trp 72. The project involves extensive NMR experimentation aimed at identifying and assigning the complete kringles' spectra and at characterizing the conformational dynamics including H-exchange and ligand on-off binding kinetics. Since kringles are present in other blood plasma proteins such as prothrombin, the tissue plasminogen activator, fibronectin and haptoglobin, and in urokinase, a kidney plasminogen activator, it is clear that the proposed studies have implications in the understanding of key kringle-mediated protein-protein interactions in haemostasis. In this context, current efforts to develop emergency thrombosis therapy via bio-engineered plasminogen activators ought to be greatly assisted if the cloned kringles carried structural components which enhance their binding affinity to the fibrin clot. Furthermore, the proposed studies have direct bearing in the design of antifibrinolytic drugs aimed at promoting healing in the treatment of haemorragic disorders.

建议继续研究构象和动态 纤溶酶原（92,000 Mr）的特性，重点是了解 纤溶酶原重链的结构、功能和生化调节 链，特别是它的五个 kringles。 纤溶酶原 kringles 为 10,000 Mr 结构域和折叠域受三个 Cys-Cys 桥的约束；他们 负责将纤溶酶原锚定在纤维蛋白和血栓上，并且 可能介导与 α2-抗纤溶酶和与 纤溶酶原预激活肽（止血控制因子）。 这些 相互作用被认为涉及赖氨酸结合位点（LBS），已知 被传送到 kringle 1 (K1)、K2，并且很可能被 K5 传送。 K1、K4、K5 通过纤溶酶原的蛋白水解裂解分离组合的 K2+3 从人血浆中提取。 然后对 kringles 进行一维 1H2O 中 300 和 600 MHz（1H 频率）的 2-D NMR 光谱 和2H2O中。 将特别强调系统地 表征配体药物的结构/空间要求和 含有 C 端 L-Lys 的短模型合成多肽链。 从 Overhauser 对 K4 的研究和其他化学证据表明， LBS 由 Cys51 -Cys75 环构成，并涉及极性残基 Asp57 和 Arg71，以及 Phe 64 和 Trp 72 的芳香族侧链。 项目涉及广泛的核磁共振实验，旨在识别和 分配完整的克林格斯光谱并表征 构象动力学，包括 H 交换和配体开关结合 动力学。 由于 kringles 存在于其他血浆蛋白中，例如 如凝血酶原、组织纤溶酶原激活剂、纤连蛋白和 触珠蛋白，以及尿激酶（一种肾脏纤溶酶原激活剂），很明显 拟议的研究对理解关键 kringle 介导的止血中蛋白质-蛋白质相互作用。 在这个 背景下，目前开发紧急血栓治疗的努力 如果生物工程纤溶酶原激活剂应该得到很大帮助 克隆的三环携带增强其结合的结构成分 与纤维蛋白凝块的亲和力。 此外，拟议的研究直接 参与旨在促进愈合的抗纤维蛋白溶解药物的设计 用于治疗血液疾病。