ABSTRACT
Raso, S.W., Clark, P.L., Haase-Pettingell, C., King, J. and Thomas, G.J., Jr. (2001) J. Mol. Biol., 307, 899-911.
Cysteine sulfhydryl environments of phage P22 tailspike trimer: analysis of raman S-H markers of Cys - > Ser mutant proteins.
Very little is known about the character or functional relevance of hydrogen-bonded cysteine thiols in proteins. In this report, we combine site-specific mutagenesis and Raman spectroscopy to probe several distinct cysteine environments in a large homotrimeric protein. The 666-residue subunit of the Salmonella typhimurium bacteriophage P22 tailspike contains eight cysteines widely distributed through the sequence (Cys 169, Cys 267, Cys 287, Cys 290, Cys 458, Cys 496, Cys 613 and Cys 635). Although no Cys forms intra- or inter-subunit disulfides in the native state, a subset is involved in transient SÐS bond formation during folding and assembly. Tailspike cysteines are also characterized by an extraordinary Raman SÐH band complex (2500-2600 cm-1 interval) indicative of diverse SÐH hydrogen-bonding interactions. Because the Raman SÐH band is a unique and sensitive probe of the local SÐH environment, it is of interest to resolve specific Cys contributions to the complex Raman band of the native protein. We have elucidated specific hydrogen-bonding environments of all eight tailspike sulfhydryls by constructing single-site (Cys à Ser) mutants and systematically analyzing their respective Raman signatures. Importantly, the recombinant proteins, once folded, are shown by Raman amide bands and activity assays to be structurally and functionally indistinguishable from tailspikes assembled in P22-infected Salmonella. We find that: (i) Cys 613 forms the strongest SÐHáááX bond of the native tailspike, stronger by far than any heretofore observed for a protein. (ii) Robust SÐHáááX bonding is also indicated for Cys 267, Cys 287 and Cys 458. (iii) Moderately strong hydrogen bonding is found for Cys 169 and Cys 635. (iv) Raman markers of Cys 290 and Cys 496 indicate the weakest hydrogen bonds. (v) Remarkably, Cys 287 contributes two Raman SÐH markers, indicating the population of two distinct hydrogen-bonding states. (vi) The sum of the SÐH Raman signatures of all eight mutants accurately reproduces the composite Raman band of the wild-type tailspike, implying that sulfhydryl environments in the native structure are not interdependent. SDS denaturation of the tailspike converts all sulfhydryls to a similar hydrogen-bonding state, as expected. This study represents the first detailed assessment of local SÐH hydrogen bonding in a native protein and provides information not obtainable by any other structural probe. The results provide a basis for assessing the roles of individual cysteines in the novel folding/assembly pathway of the P22 tailspike. This method should be applicable to a wide range of cysteine-containing proteins, including viral structural proteins, thiol oxidoreductases and Cys ligand-binding factors.
