Monitoring protein interactions in the living cell through the fluorescence decays of the cyan fluorescent protein.
Chemphyschem
; 7(7): 1442-54, 2006 Jul 17.
Article
en En
| MEDLINE
| ID: mdl-16739159
ABSTRACT
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.
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Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Proteínas
/
Transferencia Resonante de Energía de Fluorescencia
/
Proteínas Fluorescentes Verdes
/
Microscopía Fluorescente
Límite:
Humans
Idioma:
En
Revista:
Chemphyschem
Asunto de la revista:
BIOFISICA
/
QUIMICA
Año:
2006
Tipo del documento:
Article
País de afiliación:
Francia