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Development of a FRET Assay for Monitoring of HIV gp41 Core Disruption
journal contribution
posted on 2007-08-31, 00:00 authored by Yang Xu, Mark S. Hixon, Philip E. Dawson, Kim D. JandaThe fusogenic core assembly of human immunodeficiency virus type 1 (HIV-1) fusion protein gp41 is
a critical transformation for viral entry. Molecules that are able to intercept this process are of great
therapeutic value as HIV-1 fusion inhibitors. In the search for such molecules, assay systems that can be
adapted to high-throughput screens are valuable. Given that gp41 fusogenic transformation is characterized
by the hexameric association of heptads located at the N and C terminal regions of the protein ectodomain,
the corresponding heptad peptides (CHR and NHR), known to form the six-helix bundle core of gp41
fusion active form, are potentially useful in developing a fluorescence resonance energy transfer (FRET)
system for identification of HIV fusion inhibitors. We demonstrate that by strategically placing two FRET
probes on these two peptides, we are able to monitor the intermolecular co-association by fluorescence
quenching between the fluorescence donor and acceptor. The utility of the system is that it should be
adaptable to high-throughput screening (HTS) toward peptide or small-molecule HIV fusion inhibitors
targeting the gp41 core. Herein, we report the design, synthesis, and development of a N- and C- terminal
peptide FRET pair for screening of gp41 six-helix bundle disruption.
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CHRterminal peptide FRET pairgp 41 fusionprotein ectodomainFRET Assayassay systemsHTSheptad peptidesgp 41 coreC terminal regionsimmunodeficiency virus type 1fluorescence quenchingfluorescence donorHIV gp 41 Core DisruptionThe fusogenic core assemblyfluorescence resonance energy transfergp 41 fusogenic transformationNHRFRET probeshexameric associationHIV fusion inhibitorsfusion inhibitors
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