Mimicking the pre-hairpin intermediate of gp41
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Abstract
A novel screening platform for potential retroviral fusion inhibitors on the basis of fully
functional membrane‐anchored coiled coil lipopeptide receptors has been established. The
work comprises the scrutiny of lateral organization of functional lipids in phase separated
bilayers and an in‐depth investigation of the biophysical properties of lipopeptide‐based
receptors. Lateral sorting of lipids was detected by the recognition of streptavidin of
biotinylated lipids in phase separated bilayers and by nanoscopic patterns in mixed
fluorocarbon / hydrocarbon lipid bilayers, employing temperature controlled atomic force
nmicroscopy (AFM) as a versatile characterization method. Particular features of fluorocarbon
bilayers were additionally investigated in great detail by means of ellipsometry and ATR‐IR
spectroscopy. Lipopeptide‐receptors were synthesized on the basis of a robust and reliable
in situ coupling reaction by coupling terminal cysteine modified receptor‐peptides to a
maleimide functionalized lipid bilayer. Receptor functionality of the lipopeptides was
visualized by specific binding of vesicles and nanoparticles tracked by a multiplicity of
characterization methods, such as AFM, ellipsometry, CLSM and fluorescence spectroscopy.
Finally, in situ coupling of viral peptides, originating from the fusion protein of HIV resulted
in a mimic of the pre‐hairpin intermediate of gp41. Structural analysis of N36‐lipopepides by
means of CD‐spectroscopy in combination with FT‐IR spectroscopy revealed a coiled coil
assembly of lipopeptides, which render the aggregates fully functional receptors for
potent
fusion inhibitors. Thereby, reversible inhibitor binding of T20 and the corresponding C‐
peptides was detected by AFM and ellipsometry, rendering coiled coil lipopeptides a new
promising technique for screening of retroviral fusion inhibitors.