Development of a FRET assay for monitoring of HIV gp41 core disruption.
The 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.
Xu
Yang
Y
Department of Chemistry and Immunology and The Skaggs Institute for Chemical Biology and Worm Institute for Research and Medicine (WIRM), The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Hixon
Mark S
MS
Dawson
Philip E
PE
Janda
Kim D
KD
D000595:Amino Acid Sequence; D055592:Biophysical Phenomena; D001703:Biophysics; D002942:Circular Dichroism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D018698:Glutamic Acid / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry; D015700:HIV Envelope Protein gp41 / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021241:Spectrometry, Mass, Electrospray Ionization
D015700:HIV Envelope Protein gp41; D010455:Peptides; D018698:Glutamic Acid; D019791:Guanidine