A protease assay for two-photon crosscorrelation and FRET analysis based solely on fluorescent proteins

Research output: Contribution to journalResearch articleContributedpeer-review

Contributors

  • Tobias Kohl - (Author)
  • Katrin G. Heinze - (Author)
  • Rene Kuhlemann - , DIREVO Biotech AG (Author)
  • Andre Koltermann - , DIREVO Biotech AG (Author)
  • Petra Schwille - , Chair of Biophysics (Author)

Abstract

GFP and the red fluorescent protein, DsRed, have been combined to design a protease assay that allows not only for fluorescence resonance energy transfer (FRET) studies but also for dual-color crosscorrelation analysis, a single-molecule-based method that selectively probes the concomitant movement of two distinct tags. The measurement principle is based on a spectrally resolved detection of single molecules diffusing in and out of a diffraction-limited laser focus. Double-labeled substrate molecules are separated into two single-labeled products by specific cleavage at a protease cleavage site between the two flanking tags, DsRed and GFP, thus disrupting joint fluctuations in the two detection channels and terminating FRET between the two labels. In contrast to enzyme assays based solely on FRET, this method of dual-color crosscorrelation is not limited to a certain range of distances between the fluorophores and is much more versatile with respect to possible substrate design. To simplify the measurement setup, two-photon excitation was used, allowing for simultaneous excitation of both tags with a single infrared laser wavelength. The general concept was experimentally verified with a GFP-peptide-DsRed construct containing the cleavage site for tobacco etch virus protease. Two-photon excitation in the infrared and the use of cloneable tags make this assay easily adaptable to intracellular applications. Moreover, the combination of FRET and crosscorrelation analysis in a single-molecule-based approach promises exciting perspectives for miniaturized high-throughput screening based on fluorescence spectroscopy.

Details

Original languageEnglish
Pages (from-to)12161-12166
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America : PNAS
Volume99
Issue number19
Publication statusPublished - 17 Sept 2002
Peer-reviewedYes

External IDs

PubMed 12209012

Keywords

ASJC Scopus subject areas