SALT BRIDGE CONVERSION TO AMIDE BONDS WITH C2N2

我们已经证明了这种气体试剂，C2N，可以用来引入 通过将某些盐桥和羧基转化为蛋白质- 将羧酸盐对转化为共价键。我们打算定义(1)类型 做出反应的功能，(2)做出反应的组的环境 反应，以及(3)对于选定的蛋白质，残基的特定特性 牵涉其中。这似乎是唯一一种特定的蛋白质修饰试剂 用于成对的官能团。 对C2N2修改的盐桥类型的明确定义允许 适当使用C2N2成为定位盐桥和 羧基-羧酸盐相互作用。C2N2作为一种新型燃料的成功开发 工具，这一提议的目标，将允许采取方法来 目前正在调查的远程目标。自.以来 绝大多数蛋白质仍未被X射线确定 结晶学，它将有可能(1)扩大我们对 离子对的重要性，(2)发展C_2N_2共价连接的用途 盐桥联到结合蛋白的配体，(3)探索 使用C2N2来共价连接相关的亚基，以便 完成多肽在完整蛋白质中的组装 多肽，(4)测试瞬变盐桥 折叠/展开过程，(5)故意修饰蛋白质的“非- 基本的“盐桥”，从而增加稳定性，(6)至 评估盐桥在蛋白质稳定性中的作用 嗜热有机体。 总体策略是将C2N2处理的条件([C2N2]， PH、T、T、特殊和一般盐效应...)的变化对蛋白质的影响 官能度(酶活性，氧结合和协作性，等电点， 多分散性，官能团修饰...)。有了这些关系 将观察到的交联链与已知的盐进行比较 桥梁将会搭建起来。布鲁克海文蛋白质数据库或剑桥数据 BANK将用于帮助确定C2N2反应性盐的共同特征 大桥。了解结构组件的作用是基本的 致力于解决结构和运作问题的任何努力，尤其是 与目前设计新蛋白质设计的努力相关。

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.