Abstract
Genetically encoded Ca(2+) indicators (GECI) are important for the measurement of Ca(2+) in vivo. GCaMP2, a widely-used GECI, has recently been iteratively improved. Among the improved variants, GCaMP3 exhibits significantly better fluorescent intensity. In this study, we developed a new GECI called GCaMPJ and determined the crystal structures of GCaMP3 and GCaMPJ. GCaMPJ has a 1.5-fold increase in fluorescence and 1.3-fold increase in calcium affinity over GCaMP3. Upon Ca(2+) binding, GCaMP3 exhibits both monomeric and dimeric forms. The structural superposition of these two forms reveals the role of Arg-376 in improving monomer performance. However, GCaMPJ seldom forms dimers under conditions similar to GCaMP3. St ructural and mutagenesis studies on Tyr-380 confirmed its importance in blocking the cpEGFP β-barrel holes. Our study proposes an efficient tool for mapping Ca(2+) signals in intact organs to facilitate the further improvement of GCaMP sensors.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Calcium / chemistry
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Calcium / metabolism
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Calmodulin / chemistry
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Calmodulin / genetics
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Calmodulin / metabolism
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Crystallography, X-Ray
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Dimerization
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Green Fluorescent Proteins / chemistry
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / metabolism
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Histidine / chemistry
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Histidine / genetics
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Histidine / metabolism
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Hydrogen-Ion Concentration
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Myosin-Light-Chain Kinase / chemistry
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Myosin-Light-Chain Kinase / genetics
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Myosin-Light-Chain Kinase / metabolism
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Peptide Fragments / chemistry
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Peptide Fragments / genetics
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Peptide Fragments / metabolism
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Protein Structure, Tertiary
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Recombinant Fusion Proteins / biosynthesis
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Recombinant Fusion Proteins / chemistry*
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Recombinant Fusion Proteins / genetics
Substances
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Calmodulin
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M13 protein (myosin light-chain kinase)
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Peptide Fragments
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Recombinant Fusion Proteins
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enhanced green fluorescent protein
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Green Fluorescent Proteins
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polyhistidine
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Histidine
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Myosin-Light-Chain Kinase
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Calcium