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New HIV-protease assays applying self-quenching peptide substrates in combination with time-resolved fluorescence single-molecule spectroscopy
Authors:Thorsten Martin Staudt  Hans-Georg Kräusslich
Institution:1. High Resolution Optical Microscopy , German Cancer Research Center , Im Neuenheimer Feld 280, 69120 Heidelberg, Germany;2. Department of Virology , Hygiene Institute , Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
Abstract:This work describes the optimization and adoption of an assay system for the Human Immunodeficiency Virus (HIV)-protease, whose inhibition plays a central role in HIV therapy. The HIV-protease, which is an essential enzyme during viral maturation, has a specific cleavage site of eight amino acid residues (SQNY*PIV). Adding two amino acid residues at the N-terminus and enclosing the resulting sequence by a dye-labelled lysine residue and a tryptophan residue leads to the substrate (K(dye)CGSQNY*PIVW) in which the fluorescence of the fluorophore is efficiently quenched by the intrinsic tryptophan due to a photoinduced electron transfer reaction. After cleavage of the substrate by the target enzyme, the dye and the tryptophan residue are separated, effecting a significant increase in fluorescence intensity. Measuring the fluorescence versus time enables an online-monitoring of the enzyme activity. With this method, a HIV-PR concentration of 10?9?M is detectable within minutes, which is comparable with commercially available assays using doubly labelled substrates based on a fluorescence resonance energy transfer. We were able to further increase the sensitivity to the subnanomolar range by using confocal single-molecule spectroscopy.
Keywords:Proteolytic enzymes  HIV-protease  Photoinduced electron transfer  Single-molecule spectroscopy
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