Circularly permuted green fluorescent proteins engineered to sense Ca2+.
To visualize Ca(2+)-dependent protein-protein interactions in living cells by fluorescence readouts, we used a circularly permuted green fluorescent protein (cpGFP), in which the amino and carboxyl portions had been interchanged and reconnected by a short spacer between the original termini. The cpGFP was fused to calmodulin and its target peptide, M13. The chimeric protein, which we have named "pericam," was fluorescent and its spectral properties changed reversibly with the amount of Ca(2+), probably because of the interaction between calmodulin and M13 leading to an alteration of the environment surrounding the chromophore. Three types of pericam were obtained by mutating several amino acids adjacent to the chromophore. Of these, "flash-pericam" became brighter with Ca(2+), whereas "inverse-pericam" dimmed. On the other hand, "ratiometric-pericam" had an excitation wavelength changing in a Ca(2+)-dependent manner. All of the pericams expressed in HeLa cells were able to monitor free Ca(2+) dynamics, such as Ca(2+) oscillations in the cytosol and the nucleus. Ca(2+) imaging using high-speed confocal line-scanning microscopy and a flash-pericam allowed to detect the free propagation of Ca(2+) ions across the nuclear envelope. Then, free Ca(2+) concentrations in the nucleus and mitochondria were simultaneously measured by using ratiometric-pericams having appropriate localization signals, revealing that extra-mitochondrial Ca(2+) transients caused rapid changes in the concentration of mitochondrial Ca(2+). Finally, a "split-pericam" was made by deleting the linker in the flash-pericam. The Ca(2+)-dependent interaction between calmodulin and M13 in HeLa cells was monitored by the association of the two halves of GFP, neither of which was fluorescent by itself.
Nagai
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Laboratory for Cell Function and Dynamics, Advanced Technology Development Center, Brain Science Institute, Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
Sawano
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A
Park
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ES
Miyawaki
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D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism; D002413:Cations, Divalent; D019281:Dimerization; D015870:Gene Expression; D017930:Genes, Reporter; D005818:Genetic Engineering; D014644:Genetic Variation; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000235:genetics / Q000378:metabolism; D009685:Nuclear Envelope / Q000378:metabolism / Q000502:physiology; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010539:Permeability; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D002147:Calmodulin; D002413:Cations, Divalent; D008164:Luminescent Proteins; C107660:M13 protein (myosin light-chain kinase); D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D009219:Myosin-Light-Chain Kinase; D002118:Calcium