Detection in living cells of Ca2+-dependent changes in the fluorescence emission of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence. A new class of fluorescent indicators.
J Biol Chem
; 272(20): 13270-4, 1997 May 16.
Article
en En
| MEDLINE
| ID: mdl-9148946
We have designed a novel fluorescent indicator composed of two green fluorescent protein variants joined by the calmodulin-binding domain from smooth muscle myosin light chain kinase. When (Ca2+)4-calmodulin is bound to the indicator (Kd = 0.4 nM), fluorescence resonance energy transfer between the two fluorophores is attenuated; the ratio of the fluorescence intensity measured at 505 nm to the intensity measured at 440 nm decreases 6-fold. Images of microinjected living cells demonstrate that emission ratios can be used to monitor spatio-temporal changes in the fluorescence of the indicator. Changes in indicator fluorescence in these cells are coupled with no discernible lag (<1 s) to changes in the cytosolic free Ca2+ ion concentration, ranging from below 50 nM to approximately 1 microM. This observation suggests that the activity of a calmodulin target with a typical 1 nM affinity for (Ca2+)4-calmodulin is responsive to changes in the intracellular Ca2+ concentration over the physiological range. It is likely that the indicator we describe can be modified to detect the levels of ligands and proteins in the cell other than calmodulin.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas de Unión a Calmodulina
/
Calcio
/
Colorantes Fluorescentes
Tipo de estudio:
Diagnostic_studies
Límite:
Humans
Idioma:
En
Revista:
J Biol Chem
Año:
1997
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Estados Unidos