Raso S W, Clark P L, Haase-Pettingell C, King J, Thomas G J
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
J Mol Biol. 2001 Mar 30;307(3):899-911. doi: 10.1006/jmbi.2001.4476.
Very little is known about the character or functional relevance of hydrogen-bonded cysteine sulfhydryl (S-H) groups in proteins. The Raman S-H band is a unique and sensitive probe of the local S-H environment. Here, we report the use of Raman spectroscopy combined with site-specific mutagenesis to document the existence of five distinguishable hydrogen-bonded states of buried cysteine sulfhydryl groups in a native protein. The 666 residue subunit of the Salmonella typhimurium bacteriophage P22 tailspike contains eight cysteine residues distributed through the elongated structure. The tailspike cysteine residues display an unusual Raman S-H band complex (2500-2600 cm(-1) interval) indicative of diverse S-H hydrogen-bonding interactions in the native trimeric structure. To resolve specific Cys contributions to the complex Raman band we characterized a set of tailspike proteins with each cysteine replaced by a serine. The mutant proteins, once folded, were structurally and functionally indistinguishable from wild-type tailspikes, except for their Raman S-H signatures. Comparison of the Raman spectra of the mutant and wild-type proteins reveals the following hydrogen-bond classes for cysteine sulfhydryl groups. (i) Cys613 forms the strongest S-H...X bond of the tailspike, stronger than any heretofore observed for a protein. (ii) Cys267, Cys287 and Cys458 form robust S-H...X bonds. (iii) Moderate S-H...X bonding occurs for Cys169 and Cys635. (iv) Cys290 and Cys496 form weak hydrogen bonds. (v) It is remarkable that Cys287 contributes two Raman S-H markers, indicating the population of two distinct hydrogen-bonding states. The sum of the S-H Raman signatures of all eight mutants accurately reproduces the composite Raman band of the wild-type tailspike. The diverse cysteine states may be an outcome of the folding and assembly pathway of the tailspike, which though lacking disulfide bonds in the native state, utilizes transient disulfide bonds in the maturation pathway. This Raman study represents the first detailed assessment of local S-H hydrogen bonding in a native protein and provides information not obtainable directly by other structural probes. The method employed here should be applicable to a wide range of cysteine-containing proteins.
关于蛋白质中氢键连接的半胱氨酸巯基（S-H）基团的特性或功能相关性，人们了解得非常少。拉曼S-H谱带是局部S-H环境的独特且灵敏的探针。在此，我们报告结合拉曼光谱与位点特异性诱变来证明天然蛋白质中埋藏的半胱氨酸巯基存在五种可区分的氢键连接状态。鼠伤寒沙门氏菌噬菌体P22尾刺的666个残基亚基包含八个半胱氨酸残基，分布在细长结构中。尾刺半胱氨酸残基呈现出异常的拉曼S-H谱带复合体（2500 - 2600 cm(-1)区间），表明在天然三聚体结构中存在多样的S-H氢键相互作用。为了确定特定半胱氨酸对复杂拉曼谱带的贡献，我们对一组尾刺蛋白进行了表征，其中每个半胱氨酸都被丝氨酸取代。突变蛋白一旦折叠，在结构和功能上与野生型尾刺无法区分，只是它们的拉曼S-H特征不同。突变蛋白和野生型蛋白的拉曼光谱比较揭示了半胱氨酸巯基的以下氢键类别。（i）Cys613形成尾刺中最强的S-H...X键，比迄今在蛋白质中观察到的任何键都强。（ii）Cys267、Cys287和Cys458形成稳固的S-H...X键。（iii）Cys169和Cys635形成中等强度的S-H...X键。（iv）Cys290和Cys496形成弱氢键。（v）值得注意的是，Cys287贡献了两个拉曼S-H标记，表明存在两种不同的氢键连接状态。所有八个突变体的S-H拉曼特征总和准确地再现了野生型尾刺的复合拉曼谱带。多样的半胱氨酸状态可能是尾刺折叠和组装途径的结果，尾刺在天然状态下虽然缺乏二硫键，但在成熟途径中利用了瞬时二硫键。这项拉曼研究代表了对天然蛋白质中局部S-H氢键的首次详细评估，并提供了其他结构探针无法直接获得的信息。这里采用的方法应该适用于广泛的含半胱氨酸蛋白质。
