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SYBP: Biophotonik

SYBP 4: Postersitzung

SYBP 4.9: Poster

Donnerstag, 7. März 2002, 17:45–20:00, Schloss

Monitoring Protease Activity on a Single Molecule Scale using Two-Photon Cross-Correlation and FRET Analysis — •Katrin G. Heinze¹, Tobias Kohl¹, Rene Kuhlemann², Andre Koltermann², and Petra Schwille¹ — ¹Max-Planck-Institute for biophys. Chemistry, Experimental Biophysics Group, D-37077 Göttingen — ²DIREVO Biotech AG, D-50829 Köln

We have developed an assay for quantitatively monitoring enzyme kinetics utilizing a combination of Fluorescence Two-Photon Cross-Correlation (TPCC) and Resonance Energy Transfer (FRET) analysis of fluorescent proteins on a single molecule scale. While FRET is a technique relying on the distance-dependent energy transfer between adjacent fluorophores, TPCC, an extension of FCS based on two-photon excitation (TPE), is independend of fluorophore position and allows for detection of the dissociation or association of molecules with spectrally different emissions. We monitored a protease digest of a fusion protein of the fluorescent proteins, rsGFP and DsRed, connected by a linker containing the target site for the Tabacco Etch Virus protease TEV. The use of two-photon excitation (TPE) conferred the added benefit of efficient excitation of both fluorophores by the same wavelength and a significant reduction of background and photobleaching. These highly sensitive online measurements of the TEV protease cleavage reaction in vitro can be extended to in vivo situations and may provide a useful model system for studying protease activity or protein degradation in biological processes such as apoptosis or viral infection.