SALT BRIDGE CONVERSION TO AMIDE BONDS WITH C2N2

使用 C2N2 盐桥转化为酰胺键

• 批准号: 3301498
• 负责人: RICHARD A DAY
• 金额: $ 6.26万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-01 至 1993-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3301498
• 关键词: SDS polyacrylamide gel electrophoresis; acidity /alkalinity; amides; beta adrenergic receptor; carbonate dehydratase; carboxyl group; carboxylate; chemical bond; chemical condensation; covalent bond; crosslink; cyanogen bromide; guanidines; hemoglobin; high performance liquid chromatography; imidazole; neurophysins; oxytocin; pancreatic ribonuclease; peptides; protein sequence; protein structure; protein structure function; radiotracer; reversed phase chromatography; thermostability

We have shown that this gaseous reagent, C2N2, can be used to introduce crosslinks into proteins by converting certain salt bridges and carboxyl- carboxylate pairs into covalent bonds. We intend to define (1) the types of functionalities that do react, (2) the environment of the groups that do react, and (3) for selected proteins the specific identities of residues involved. This appears to be the only protein modifying reagent specific for pairs of functional groups. A clear definition of the types of salt bridges modified by C2N2 allows the appropriate use of C2N2 to become a tool for locating salt bridge and carboxyl-carboxylate interactions. Successful development of C2N2 use as a tool, the objective of this proposal, will allow approaches to be made to long range objectives under investigation at the present time. Since the overwhelming majority of proteins remain undefined by X-ray crystallography, it will be possible (1) to extend our understanding of the importance of ion pairs, (2) develop the use of C2N2 to attach covalently ligands that are salt-bridged to the binding protein, (3) to explore the use of C2N2 to link subunits covalently that are associated in order to complete the assembly of polypeptides into the completed protein polypeptide, (4) to test for transient salt bridges in the folding/unfolding process, (5) to modify deliberately a protein's "non- essential" salt bridges and thereby impart increased stability, (6) to evaluate the role of salt bridges in the stability of proteins in thermophilic organisms. The overall strategy is to relate the conditions of C2N2 treatment ([C2N2], pH, T, t, specific and general salt effects...) of proteins to changes in functionality (enzymatic activity, oxygen binding and cooperativity, pI, polydispersity, functional groups modified...). With these relationships in hand a comparison of the observed cross links with the known salt bridges will be made. The Brookhaven Protein Data Base or Cambridge Data Bank will be used to help identify common features of C2N2 reactive salt bridges. The understanding of the role of structural components is basic to any effort to address structure to function problems and is particularly relevant to current efforts to engineer new protein designs.

我们已经表明，这种气态试剂C2 N2可以用来引入 通过将某些盐桥和羧基 羧基配对成共价键。 我们打算定义（1）类型 （2）发生反应的群体的环境 反应，以及（3）对于选定的蛋白质，残基的特定身份 涉案 这似乎是唯一的蛋白质修饰试剂特异性 对于成对的官能团。 C2 N2修饰的盐桥类型的明确定义允许 适当使用C2 N2，成为定位盐桥的工具， 羧基-羧基相互作用。 成功开发C2 N2作为 工具，本提案的目标，将允许采取的办法， 目前正在调查的长期目标。 以来 绝大多数的蛋白质在X射线下仍然是不确定的 晶体学，将有可能（1）扩展我们对晶体学的理解 离子对的重要性，（2）开发使用C2 N2共价连接 盐桥连到结合蛋白的配体，（3）探索 使用C2 N2共价连接缔合的亚基， 完成多肽组装成完整的蛋白质 多肽，（4）以测试瞬时盐桥在 折叠/解折叠过程，（5）故意修饰蛋白质的“非- 必要的“盐桥”，从而增强稳定性，（6） 评估盐桥在蛋白质稳定性中的作用， 嗜热生物 总体策略是将C2 N2治疗的条件（[C2 N2]， pH、T、t、特定和一般盐效应.）蛋白质的变化 功能性（酶活性，氧结合和协同性，pI， 多分散性、官能团改性.）。 有了这些关系 在手中的比较观察到的交联与已知的盐 桥梁将被建造。 布鲁克海文蛋白质数据库或剑桥数据 Bank将用于帮助识别C2 N2反应性盐的共同特征 桥梁. 对结构部件作用的理解是基本的 任何努力，以解决结构功能的问题，特别是 与目前设计新蛋白质的努力有关。