Determination of the disulfide bond arrangement of dengue virus NS1 protein.
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.
Wallis
Tristan P
TP
Department of Microbiology and Parasitology, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland 4072, Australia.
Huang
Chang-Yi
CY
Nimkar
Subodh B
SB
Young
Paul R
PR
Gorman
Jeffrey J
JJ
D000595:Amino Acid Sequence; D003553:Cystine; D004220:Disulfides; D010450:Endopeptidases; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin; D017361:Viral Nonstructural Proteins / Q000737:chemistry*
D004220:Disulfides; C079340:NS1 protein, Dengue virus type 2; D010446:Peptide Fragments; D017361:Viral Nonstructural Proteins; D003553:Cystine; D010450:Endopeptidases; D014357:Trypsin