CONFORMATIONAL STUDIES OF PROTEINS IN CONTACT ACTIVATION

本项目的长期目标是了解 负责表达这四种基因的生物活性 参与血液凝固接触阶段的蛋白质。 的 它们相互作用的构象和结构方面将是 使用各种生物物理方法，如圆二色性， 溶剂微扰，紫外差光谱，光散射， 沉淀、化学修饰、分子模型构建和 蛋白质二级结构预测 具体而言，目标将是：（1）因子XII中的肽序列 合成已知参与表面结合的化合物。 这些肽，其对应于图1中的因子Xiii-28和因子XII 134 -153。 完整的因子XII将被氨基酸取代修饰， 特殊化学修饰 系统动力学和构象 将进行分析，以确定各种形式的修改 将通过以下方式影响因子XII活化的抑制： 带负电荷的表面 (2)含有GHKHER的肽和 已知参与表面结合的HMWK中的HGLGHGH序列 将被合成。 已知HMWK中的这些参与了 将合成表面结合。 这些独特的序列， 对应于完整HMWK的HMWK 426 -431、HMWK 441 -447和HMWK 451 -457 将通过氨基酸取代和特定化学物质进行修饰 修改. 系统的动力学和构象分析将是 进行调查如何在电荷和氨基酸的各种变化 取代将影响肽抑制HMWK的能力 将合成与PK和因子Xi结合的多肽。 这些肽， 对应于完整HMWK中的HMWK 556 -595和HMWK 556 -613， 通过氨基酸取代和化学修饰进行修饰。 系统 将进行动力学和构象分析以研究如何 肽的各种修饰方式将影响 抑制HMWK与两种酶原的结合。 (4)最后，使用 化学交联剂，与合成肽结合， HMWK 556 -595和HMWK 556 -613，以建立和定义HMWK PK和因子Xi中的结合位点。

The long term goal of this project is to understand the mechanism that are responsible for the expression of biological activities of each of the four proteins involved in the contact phase of blood coagulation. The conformational nad structural aspects of their interactions will be investigated using various biophysical methods, such as circular dichroism, solvent perturbation, uv difference spectroscopy, light scattering, sedimentation, chemical modification, molecular model building and prediction of protein secondary structure. Specifically, the objectives will be: (1) Peptide sequences in factor XII which are known to be involved in surface binding will be synthesized. These peptides, which correspond to factor Xiii-28 and factor XII134-153 of the intact factor XII will be modified by amino acid substitution and specific chemical modifications. Systematic kinetic and conformational analysis will be undertaken to determine how various forms of modifications of the peptides will affect the inhibition of factor XII activation by negatively charged surfaces. (2) Peptides containing the GHKHER and HGLGHGH sequences in HMWK which are known to be involved in surface binding will be synthesized. These in HMWK which are known to be involved in surface binding will be synthesized. These unique sequences, which correspond to HMWK426-431, HMWK441-447 and HMWK451-457 of the intact HMWK will be modified by amino acid substitution and specific chemical modifications. Systematic kinetic and conformational analysis will be conducted to investigate how various alterations in charges and amino acid substitutions will affect the ability of the peptides to inhibit HMWK binding to PK and factor XI will be synthesized. These peptides, which correspond to HMWK556-595 and HMWK556-613 in the intact HMWK will be modified by amino acid substitution and chemical modifications. Systematic kinetic and conformational analysis will be undertaken to investigate how various ways of modifications of the peptides will affect the ability to inhibit the binding of HMWK to the two zymogen. (4) Finally, to use chemical crosslinking reagent, in conjunction with the synthetic peptides, HMWK556-595 and HMWK556-613, in order to establish and define the HMWK binding site in PK and factor XI.