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

  • Raso S
  • Clark P
  • Haase-Pettingell C
 et al. 
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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.

Author-supplied keywords

  • Amino Acid Substitution
  • Amino Acid Substitution: genetics
  • Bacteriophage P22
  • Bacteriophage P22: chemistry
  • Bacteriophage P22: genetics
  • Circular Dichroism
  • Crystallography
  • Cysteine
  • Cysteine: genetics
  • Cysteine: metabolism
  • Disulfides
  • Disulfides: chemistry
  • Disulfides: metabolism
  • Fluorescence
  • Glycoside Hydrolases
  • Glycoside Hydrolases: chemistry
  • Glycoside Hydrolases: genetics
  • Glycoside Hydrolases: metabolism
  • Hydrogen Bonding
  • Mass Spectrometry
  • Models
  • Molecular
  • Mutation
  • Mutation: genetics
  • Protein Conformation
  • Protein Folding
  • Raman
  • Recombinant Proteins
  • Recombinant Proteins: chemistry
  • Recombinant Proteins: genetics
  • Recombinant Proteins: metabolism
  • Salmonella typhimurium
  • Salmonella typhimurium: virology
  • Serine
  • Serine: genetics
  • Serine: metabolism
  • Spectrometry
  • Spectrum Analysis
  • Viral Tail Proteins
  • Viral Tail Proteins: chemistry
  • Viral Tail Proteins: genetics
  • Viral Tail Proteins: metabolism
  • X-Ray

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  • S W Raso

  • P L Clark

  • C Haase-Pettingell

  • J King

  • G J Thomas

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