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.
Department of Microbiology and Parasitology, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland 4072, Australia.
Tristan P
TP
Wallis
Chang-Yi
CY
Huang
Subodh B
SB
Nimkar
Paul R
PR
Young
Jeffrey J
JJ
Gorman
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