Determination of the Disulfide Bond Arrangement of Dengue Virus NS1 Protein

• Published in: The Journal of biological chemistry 2004-05, Vol.279 (20), p.20729-20741
• Main Authors: Wallis, Tristan P, Huang, Chang-Yi, Nimkar, Subodh B, Young, Paul R, Gorman, Jeffrey J
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Cystine; Dengue virus; Disulfides; Endopeptidases; Molecular Sequence Data; Peptide Fragments - chemistry; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Trypsin; Viral Nonstructural Proteins - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M312907200

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.