FLUORESCENCE ENERGY TRANSFER-SENSITIZED PHOTOBLEACHING OF A FLUORESCENT LABEL AS A TOOL TO STUDY DONOR-ACCEPTOR DISTANCE DISTRIBUTIONS AND DYNAMICS IN PROTEIN ASSEMBLIES - STUDIES OF A COMPLEX OF BIOTINYLATED IGM WITH STREPTAVIDIN AND AGGREGATES OF CONCANAVALIN-A

A photokinetic method of detection of fluorescence resonance energy tr ansfer (FRET) between special fluorescent labels is applied to study t ime-averaged spatial distribution of labeled proteins in protein assem blies. Prolonged irradiation of a sample at the absorption maximum of the energy donor label initiates FRET-sensitized fluorescence photoble aching of the energy acceptor label, which was monitored by steady-sta te fluorimetric measurements. Kinetics of the acceptor photobleaching and kinetics of decreasing the efficiency of FRET from donors to unble ached accepters were determined. The FRET efficiency was found from me asuring sensitization of acceptor fluoroscence. Analysis of the photok inetic data permits to estimate the time-averaged distribution of acce pters on donor-acceptor distances in the range of characteristic dista nces of FRET. Dynamic processes influencing donor-acceptor distances c an be also investigated by the method. Application of the method is de monstrated by the studies of a complex of biotinylated IgM with strept avidin and aggregates composed of concanavalin A and sodium dodecyl su lphate. A new thiadicarbocyanine dye was used as the acceptor label, R -phycoerythrin and tetramethylrhodamine isothiocyanate were the donor labels. Ln the IgM-streptavidin complex, 16% of accepters most contrib uted to FRET provided 90% of FRET efficiency, whereas accepters made a bout the same time-averaged contribution to FRET in the concanavalin A aggregates. (C) 1997 Elsevier Science S.A.