Application of fluorescence resonance energy transfer to the GroEL-GroES chaperonin reaction

Fluorescence resonance energy transfer (FRET) is a sensitive and flexible m ethod for studying protein-protein interactions. Here it is applied to the GroEL-GroES chaperonin system to examine the ATP-driven dynamics that under lie protein folding by this chaperone. Relying on the known structures of G roEL and GroES, sites for attachment of fluorescent probes are designed int o the sequence of both proteins. Because these sites are brought close in s pace when GroEL and GroES form a complex, excitation energy can pass from a donor to an acceptor chromophore by FRET. While in ideal circumstances FRE T can be used to measure distances, significant population heterogeneity in the donor-to-acceptor distances in the GroEL-GroES complex makes distance determination difficult. This is due to incomplete labeling of these large, oligomeric proteins and to their rotational symmetry. It is shown, however , that FRET can still be used to follow protein-protein interaction dynamic s even in a case such as this, where distance measurements are either not p ractical or not meaningful. In this way, the FRET signal is used as a simpl e proximity sensor to score the interaction between GroEL and GroES. Simila rly. FRET can also be used to follow interactions between GroEL and a fluor escently labeled substrate polypeptide. Thus, while knowledge of molecular structure aids enormously in the design of FRET experiments, structural inf ormation is not necessarily required if the aim is to measure the thermodyn amics or kinetics of a protein interaction event by following changes in th e binding proximity of two components. (C) 2001 Academic Press.