Distinct cysteine sulfhydryl environments detected by analysis of Raman S-H markers of Cys -> Ser mutant proteins

Very little is known about the character or functional relevance of hydroge n-bonded cysteine sulfhydryl (S-H) groups in proteins. The Raman S-H band i s a unique and sensitive probe of the local S-H environment. Here, we repor t the use of Raman spectroscopy combined with site-specific mutagenesis to document the existence of five distinguishable hydrogen-bonded states of bu ried cysteine sulfhydryl groups in a native protein. The 666 residue subuni t of the Salmonella typhimurium bacteriophage P22 tailspike contains eight cysteine residues distributed through the elongated structure. The tailspik e cvsteine residues display an unusual Raman S-H band complex (2500-2606 cm (-1) interval) indicative of diverse S-H hydrogen-bonding interactions in t he native trimeric structure. To resolve specific Cys contributions to the complex Raman band we characterized a set of tailspike proteins with each c ysteine replaced by a serine. The mutant proteins, once folded, were struct urally and functionally indistinguishable from wild-type tailspikes, except for their Raman S-III signatures. Comparison of the Raman spectra of the m utant and wildtype proteins reveals the following hydrogen-bond classes for cysteine sulfhydryl groups. (i) Cys613 forms the strongest S-H X bond of t he tailspike, stronger than any heretofore observed for a protein. (ii) Cys 267, Cys287 and Cys458 form robust S-H X bonds. (iii) Moderate S-H X bondin g occurs for Cys169 and Cys635. (iv) Cys290 and Cys496 form weak hydrogen b onds. (v) It is remarkable that Cys287 contributes two Raman S-H markers, i ndicating the population of two distinct hydrogen-bonding states. The sum o f the S-H Raman signatures of all eight mutants accurately reproduces the c omposite Raman band of the wild-type tailspike. The diverse cysteine states may be an outcome of the folding and assembly pathway of the tailspike, wh ich 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 probe s. The method employed here should be applicable to a wide range of cystein e-containing proteins. (C) 2001 Academic Press.