ArticleProceedings of the National Academy of Sciences of the United States of America2001
Circularly permuted green fluorescent proteins engineered to sense Ca2+.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2001. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 393 papers.
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Who cites it
393 citing papers in PubMed, 966 citations in OpenAlex.
- Recent developments of fluorescent proteins and biosensors driven by cross-disciplinary insights.Nature methods · 2026Review
- Genetically encoded tools for tracking metabolites in live cells.Biochemical Society transactions · 2026Review
- Calcium Buffering in Astrocytes and Its Relevance for Experimental Data Interpretation and Computational Modeling.Journal of neurochemistry · 2026Review
- Redesign of Calmodulin for Genetically Encoded ZnChemical & biomedical imaging · 2026Article
- Energetic gradients emerge in developing motor-microtubule structures.bioRxiv : the preprint server for biology · 2026Article
- Allosteric Biosensors Unravel GTPase-Effector Feedback.bioRxiv : the preprint server for biology · 2026Article
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- Fast, Bright, and Reversible Fluorescent Labeling of Rhodamine-Binding Proteins.Journal of the American Chemical Society · 2026Article
- Noise Sources and Strategies for Signal Quality Improvement in Biological Imaging: A Review Focused on Calcium and Cell Membrane Voltage Imaging.Biosensors · 2026Review
- Destabilized calcium dynamics visualized using the genetically-coded probe GCaMPJ in intact hearts of Calstabin2-null mice.Frontiers in physiology · 2026Article
- Decoding Molecular Network Dynamics in Cells: Advances in Multiplexed Live Imaging of Fluorescent Biosensors.Biosensors · 2025Review
- Bipartite Genetically Encoded Biosensors to Sense Calcium Ion Dynamics at Membrane-Membrane Contact Sites.Analytical chemistry · 2025Article
- Probing spatiotemporally organized GPCR signaling using genetically encoded molecular tools.Experimental & molecular medicine · 2025Review
- Resting CaCellular and molecular life sciences : CMLS · 2025Article
- The Multifaceted Role of Calcium Signaling in Regulated Necrosis.Biomolecules · 2025Review
- Genetically encoded biosensors of metabolic function for the study of neurodegeneration, a review and perspective.Neurophotonics · 2025Review
- Fluorescent sensors for intracellular signaling in the brain: imaging neurons, glia, and vascular cells.Neurophotonics · 2025Article
- Genetically encoded sensors illuminateIBRO neuroscience reports · 2025Article
- Split genetically encoded calcium indicators for interorganellar junctions.Proceedings of the National Academy of Sciences of the United States of America · 2025Article
- Principles and Design of Molecular Tools for Sensing and Perturbing Cell Surface Receptor Activity.Chemical reviews · 2025Review
333 more citing papers are in PubMed but not listed here.
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Authors and funding
4 authors at 2 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
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.
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