Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin.
摘要：
Important Ca2+ signals in the cytosol and organelles are often extremely localized and hard to measure. To overcome this problem we have constructed new fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations. We have dubbed these fluorescent indicators 'cameleons'. They consist of tandem fusions of a blue- or cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13, and an enhanced green- or yellow-emitting GFP. Binding of Ca2+ makes calmodulin wrap around the M13 domain, increasing the fluorescence resonance energy transfer (FRET) between the flanking GFPs. Calmodulin mutations can tune the Ca2+ affinities to measure free Ca2+ concentrations in the range 10(-8) to 10(-2) M. We have visualized free Ca2+ dynamics in the cytosol, nucleus and endoplasmic reticulum of single HeLa cells transfected with complementary DNAs encoding chimaeras bearing appropriate localization signals. Ca2+ concentration in the endoplasmic reticulum of individual cells ranged from 60 to 400 microM at rest, and 1 to 50 microM after Ca2+ mobilization. FRET is also an indicator of the reversible intermolecular association of cyan-GFP-labelled calmodulin with yellow-GFP-labelled M13. Thus FRET between GFP mutants can monitor localized Ca2+ signals and protein heterodimerization in individual live cells.
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关键词：
Amino Acid Sequence Calcium analysis chemistry Calmodulin chemistry Cytosol chemistry Energy Transfer Fluorescence Green Fluorescent Proteins Hela Cells Humans Indicators and Reagents Luminescent Proteins chemistry genetics Molecular Sequence Data Mutagenesis Myosin-Light-Chain Kinase chemistry genetics Peptide Fragments chemistry genetics Recombinant Fusion Proteins chemistry genetics Research Support Non-U.S. Gov't Research Support U.S. Gov't P.H.S.
DOI：
10.1038/42264
被引量：
年份：
1997
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