title: Dengue envelope domain III-peptide binding analysis via tryptophan fluorescence quenching assay.
abstract: In the efforts to find an anti-viral treatment for dengue, a simple tryptophan fluorescence-screening assay aimed at identifying dengue domain III envelope (EIII) protein inhibitors was developed. Residue Trp391 of EIII was used as an intrinsic probe to monitor the change in fluorescence of the tryptophan residue upon binding to a peptide. The analysis was based on the electron excitation at 280 nm and fluorescence emission at 300-400 nm of EIII, followed by quenching of fluorescence in the presence of potential peptidic inhibitors coded DS36wt, DS36opt, DN58wt and DN58opt. The present study found that the fluorescence of the recombinant EIII was quenched following the binding of DS36opt, DN58wt and DN58opt in a concentration-dependent manner. Since the λmax for emission remained unchanged, the effect was not due to a change in the environment of the tryptophan side chain. In contrast, a minimal fluorescence-quenching effect of DS36wt at 20 and 40 µM suggested that the DS36wt does not have any binding ability to EIII. This was supported by a simple native-page gel retardation assay that showed a band shift of EIII domain when incubated with DS36opt, DN58wt and DN58opt but not with DS36wt. We thus developed a low-cost and convenient spectrophotometric binding assay for the analysis of EIII-peptide interactions in a drug screening application.
authors.affiliation: Drug Design and Development Research Group, University of Malaya.
authors.forename: Aida
authors.initials: A
authors.lastname: Baharuddin
authors.forename: Asfarina
authors.initials: A
authors.lastname: Amir Hassan
authors.forename: Rozana
authors.initials: R
authors.lastname: Othman
authors.forename: Yongtao
authors.initials: Y
authors.lastname: Xu
authors.forename: Meilan
authors.initials: M
authors.lastname: Huang
authors.forename: Bimo
authors.initials: B
authors.lastname: Ario Tejo
authors.forename: Rohana
authors.initials: R
authors.lastname: Yusof
authors.forename: Noorsaadah
authors.initials: N
authors.lastname: Abdul Rahman
authors.forename: Shatrah
authors.initials: S
authors.lastname: Othman
mesh_terms: D003716:Dengue Virus / Q000378:metabolism*; D063347:Native Polyacrylamide Gel Electrophoresis; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*; D014759:Viral Envelope Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D010455:Peptides; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D014364:Tryptophan