Application of fluorescence resonance energy transfer to the GroEL-GroES chaperonin reaction.
Fluorescence resonance energy transfer (FRET) is a sensitive and flexible method for studying protein-protein interactions. Here it is applied to the GroEL-GroES chaperonin system to examine the ATP-driven dynamics that underlie protein folding by this chaperone. Relying on the known structures of GroEL and GroES, sites for attachment of fluorescent probes are designed into the sequence of both proteins. Because these sites are brought close in space when GroEL and GroES form a complex, excitation energy can pass from a donor to an acceptor chromophore by FRET. While in ideal circumstances FRET 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 dynamics even in a case such as this, where distance measurements are either not practical or not meaningful. In this way, the FRET signal is used as a simple proximity sensor to score the interaction between GroEL and GroES. Similarly, FRET can also be used to follow interactions between GroEL and a fluorescently labeled substrate polypeptide. Thus, while knowledge of molecular structure aids enormously in the design of FRET experiments, structural information is not necessarily required if the aim is to measure the thermodynamics or kinetics of a protein interaction event by following changes in the binding proximity of two components.
Rye
H S
HS
Department of Molecular Biology, Princeton University, 229 Lewis Thomas Laboratory, Princeton, New Jersey 08544, USA. hrye@molbio.princeton.edu
D018835:Chaperonin 10 / Q000737:chemistry* / Q000378:metabolism; D018834:Chaperonin 60 / Q000737:chemistry* / Q000378:metabolism; D004735:Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence / Q000379:methods*
D018835:Chaperonin 10; D018834:Chaperonin 60; D005456:Fluorescent Dyes