Determination of the disulfide bond arrangement of dengue virus NS1 protein.

  • Wallis T
  • Huang C
  • Nimkar S
 et al. 
  • 31

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Abstract

The 12 half-cystines of NS1 proteins are absolutely conserved among flaviviruses, suggesting their importance to the structure and function of these proteins. In the present study, peptides from recombinant Dengue-2 virus NS1 were produced by tryptic digestion in 100% H(2)(16)O, peptic digestion in 50% H(2)(18)O, thermolytic digestion in 50% H(2)(18)O, or combinations of these digestion conditions. Peptides were separated by size exclusion and/or reverse phase high performance liquid chromatography and examined by matrix-assisted laser desorption ionization-time of flight mass spectrometry, matrix-assisted laser desorption ionization post-source decay, and matrix-assisted laser desorption ionization tandem mass spectrometry. Where digests were performed in 50% H(2)(18)O, isotope profiles of peptide ions aided in the identification and characterization of disulfide-linked peptides. It was possible to produce two-chain peptides containing C1/C2, C3/C4, C5/C6, and C7/C12 linkages as revealed by comparison of the peptide masses before and after reduction and by post-source decay analysis. However, the remaining four half-cystines (C8, C9, C10, and C11) were located in a three-chain peptide of which one chain contained adjacent half-cystines (C9 and C10). The linkages of C8/C10 and C9/C11 were determined by tandem mass spectrometry of an in-source decay fragment ion containing C9, C10, and C11. This disulfide bond arrangement provides the basis for further refinement of flavivirus NS1 protein structural models.

Author-supplied keywords

  • Amino Acid Sequence
  • Cystine
  • Disulfides
  • Endopeptidases
  • Mass
  • Matrix-Assisted Laser Desorpti
  • Molecular Sequence Data
  • Peptide Fragments
  • Peptide Fragments: chemistry
  • Spectrometry
  • Trypsin
  • Viral Nonstructural Proteins
  • Viral Nonstructural Proteins: chemistry

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Authors

  • Tristan P Wallis

  • Chang-Yi Huang

  • Subodh B Nimkar

  • Paul R Young

  • Jeffrey J Gorman

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