Selective bridging of bis-cysteinyl residues by arsonous acid derivatives as an approach to the characterization of protein tertiary structures and folding pathways by mass spectrometry
Selective bridging of bis-cysteinyl residues by arsonous acid derivatives as an approach to the characterization of protein tertiary structures and folding pathways by mass spectrometry
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DOI：
10.1006/abio.1998.2836
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发表时间：
1998-11-15
影响因子：
2.9
通讯作者：
Glocker, MO
中科院分区：
文献类型：
--
作者：
Happersberger, HP;Przybylski, M;Glocker, MO
双半胱氨酸选择性修饰成功地应用于氧化黑素（MEL），一种芳烷酸衍生物，用于多肽和模型蛋白的三级结构研究。芳香酸修饰的肽和蛋白质可以通过质谱直接表征，例如，直接分子量测定和质谱肽图谱分别确定了化学计量和修饰位点。改良后催产素的蛋白水解消化和质谱片段分析表明，mel桥接肽衍生物与二硫键结合的大环肽在结构上是同源的。质谱分析确定了部分还原和选择性修饰的连接Cys-14和Cys-38的牛胰腺胰蛋白酶抑制剂（BPTI）的MEL修饰位点。BPTI MEL衍生物可以抵抗Lys-C和胰蛋白酶的蛋白水解，因此具有与天然BPTI类似的刚性结构。MEL表现出几个有利的特征，如：(1)交联两个紧密间隔的巯基，提供详细的三级结构信息；（ii）在水溶液和有机溶液中作为单体邻位酸具有高溶解度；（iii）单次修饰使蛋白质增加较大的质量增量；（iv）由于具有强烈的显色性，可以对衍生化进行紫外监测；(v)对蛋白质中的双巯基进行快速和特异性修饰，即使在高摩尔过量的MEL下也能形成稳定的结构，而不会产生任何副反应。所研究的MEL的物理和化学性质表明，它普遍适用于选择性双硫醇修饰，可以用于可溶性和膜蛋白的蛋白质结构-功能研究以及蛋白质折叠反应的研究。(C) 1998学术出版社。
Bis-cysteine selective modifications were successfully applied with melarsen oxide (MEL), an arsonous acid derivative, for tertiary structural studies of peptides and a model protein. The arsonous acid modified peptides and proteins were amenable to direct characterizations by mass spectrometry, e.g., direct molecular weight determinations and mass spectrometric peptide mapping that identified stoichiometry and sites of modification, respectively. Proteolytic digestion and mass spectrometric fragmentation of modified oxytocin showed that MEL-bridged peptide derivatives are structural homologues to the disulfide-bonded macrocyclic peptides. Mass spectrometric analyses determined the MEL modification site in partially reduced and selectively modified bovine pancreatic trypsin inhibitor (BPTI) bridging Cys-14 and Cys-38. The BPTI MEL derivative was resistant to proteolysis by both Lys-C and trypsin and thus represented a rigid structure like native BPTI. MEL exhibited several advantageous features such as (i) cross-linking two closely spaced thiol groups, providing detailed tertiary structure information; (ii) high solubility as monomeric ortho acid in aqueous and organic solutions; (iii) adding a relatively large mass increment to proteins upon single modification; (iv) enabling UV monitoring of the derivatization due to a strong chromophor; and (v) performing fast and specific modifications of bis-thiol groups in proteins to form stable structures without any side reactions even with a high molar excess of MEL. The investigated physical and chemical properties of MEL suggest general applicability for selective bis-thiol modifications, enabling protein structure-function studies in both soluble and membrane proteins and the study of protein-folding reactions. (C) 1998 Academic Press.