Monitoring protein interactions in the living cell through the fluorescence decays of the cyan fluorescent protein.
Using fluorescence lifetime microspectroscopy and imaging techniques, we have studied the fluorescence of cyan fluorescent protein (CFP) transiently expressed in HEK-293 cells, in the presence or absence of its fluorescence resonance energy transfer (FRET) partner, yellow fluorescent protein (YFP). When the two proteins are attached through a 27-amino-acid linker, a 33 % average efficiency of intramolecular energy transfer is accurately determined inside the cell. Additionally, we observe a systematic quenching of the CFP fluorescence with increasing levels of protein expression. This quenching cannot be accounted for by formation of the previously described dimer of GFP-related proteins, since its magnitude is unchanged when the fluorescent proteins carry the mutation A206K shown to dissociate this dimer in vitro. Even when the intracellular protein concentration largely exceeds the in vitro dissociation constant of the dimer, self-association remains undetectable, either between free proteins or intramolecularly within the CFP-YFP construct. Instead, the detailed concentration effects are satisfactorily accounted for by a model of intermolecular, concentration-dependent energy transfer, arising from molecular proximity and crowding. In the case of CFP alone, we suggest that self-quenching could result from a pseudo-homo FRET mechanism between different, spectrally shifted emissive forms of the protein. These phenomena require careful consideration in intracellular FRET studies.
Grailhe
Regis
R
Institut Pasteur, Laboratoire Recepteurs et Cognition, 25 rue du Dr. Roux, F-75015 Paris, France. regis.grailhe@pasteur.or.kr
Merola
Fabienne
F
Ridard
Jacqueline
J
Couvignou
Stephen
S
Le Poupon
Chantal
C
Changeux
Jean-Pierre
JP
Laguitton-Pasquier
Helene
H
D001426:Bacterial Proteins / Q000737:chemistry; D001703:Biophysics / Q000379:methods; D002460:Cell Line; D002627:Chemistry, Physical / Q000379:methods; D003593:Cytoplasm / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000379:methods*; D008968:Molecular Conformation; D009154:Mutation; D011506:Proteins / Q000737:chemistry*
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D011506:Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins