tailieunhanh - Báo cáo khoa học: ThermoFAD, a ThermofluorÒ-adapted flavin ad hoc detection system for protein folding and ligand binding

In living organisms, genes encoding proteins that contain flavins as a pros-thetic group constitute approximately 2–3% of the total. The fluorescence of flavin cofactors in these proteins is a property that is widely employed for biochemical characterisation. Here, we present a modified Thermofluor approach called ThermoFAD (Thermofluor -adapted flavin ad hoc detec-tion system), which simplifies identification of optimal purification and storage conditions as well as high-affinity ligands. | ỊFEBS Journal ThermoFAD a Thermofluor -adapted flavin ad hoc detection system for protein folding and ligand binding Federico Forneris Roberto Orru Daniele Bonivento Laurent R. Chiarelli and Andrea Mattevi Department of Genetics and Microbiology University of Pavia Italy Keywords flavin fluorescence screening ligand screening protein stability Thermofluoi Correspondence F. Forneris and A. Mattevi Dipartimento di Genetica e Microbiologia University di Pavia Via Ferrata 1 27100 Pavia Italy Fax 39 0382 528496 Tel 39 0382 985534 E-mail forneris@ mattevi@ Website http biocry Received 14 January 2009 revised 3 March 2009 accepted 16 March 2009 doi In living organisms genes encoding proteins that contain flavins as a prosthetic group constitute approximately 2-3 of the total. The fluorescence of flavin cofactors in these proteins is a property that is widely employed for biochemical characterisation. Here we present a modified Thermofluor approach called ThermoFAD Thermofluor -adapted flavin ad hoc detection system which simplifies identification of optimal purification and storage conditions as well as high-affinity ligands. In this technique the flavin cofactor is used as an intrinsic probe to monitor protein folding and stability taking advantage of the different fluorescent properties of flavin-containing proteins between the folded and denatured state. The main advantage of the method is that it allows a large amount of biochemical data to be obtained using very small amounts of protein sample and standard laboratory equipment. We have explored several cases that demonstrate the reliability and versatility of this technique when applied to globular flavoenzymes membrane-anchored flavoproteins and macromolecular complexes. The information gathered from ThermoFAD analysis can be very valuable for any biochemical and biophysical analysis including crystallisation. The method is likely to be .