Researchers developed circularly permuted green fluorescent proteins (cpGFPs) to sense calcium ions (Ca²⁺) in living cells. By swapping the amino and carboxyl ends of the GFP and connecting them with a short spacer, they created a protein called "pericam" that was fused with calmodulin (CaM) and a target peptide, M13. The pericam's fluorescence changed in response to Ca²⁺, likely due to the interaction between CaM and M13 altering the chromophore environment. Three variants were created by mutating amino acids near the chromophore: "flash-pericam" became brighter with Ca²⁺, "inverse-pericam" dimmed, and "ratiometric-pericam" had a Ca²⁺-dependent excitation wavelength change. These pericams could monitor free Ca²⁺ dynamics in HeLa cells, including cytosolic and nuclear Ca²⁺ oscillations. Using high-speed confocal line-scanning microscopy, they detected Ca²⁺ propagation across the nuclear envelope. Ratiometric-pericams with localization signals measured Ca²⁺ concentrations in the nucleus and mitochondria, showing that extra-mitochondrial Ca²⁺ transients caused rapid mitochondrial Ca²⁺ changes. A "split-pericam" was made by removing the linker, allowing monitoring of CaM-M13 interactions in HeLa cells. The study demonstrated that cpGFPs can be engineered to sense Ca²⁺ with high sensitivity and specificity, offering new tools for calcium imaging in living cells. The findings highlight the potential of cpGFPs as powerful tools for studying calcium signaling in cellular processes.Researchers developed circularly permuted green fluorescent proteins (cpGFPs) to sense calcium ions (Ca²⁺) in living cells. By swapping the amino and carboxyl ends of the GFP and connecting them with a short spacer, they created a protein called "pericam" that was fused with calmodulin (CaM) and a target peptide, M13. The pericam's fluorescence changed in response to Ca²⁺, likely due to the interaction between CaM and M13 altering the chromophore environment. Three variants were created by mutating amino acids near the chromophore: "flash-pericam" became brighter with Ca²⁺, "inverse-pericam" dimmed, and "ratiometric-pericam" had a Ca²⁺-dependent excitation wavelength change. These pericams could monitor free Ca²⁺ dynamics in HeLa cells, including cytosolic and nuclear Ca²⁺ oscillations. Using high-speed confocal line-scanning microscopy, they detected Ca²⁺ propagation across the nuclear envelope. Ratiometric-pericams with localization signals measured Ca²⁺ concentrations in the nucleus and mitochondria, showing that extra-mitochondrial Ca²⁺ transients caused rapid mitochondrial Ca²⁺ changes. A "split-pericam" was made by removing the linker, allowing monitoring of CaM-M13 interactions in HeLa cells. The study demonstrated that cpGFPs can be engineered to sense Ca²⁺ with high sensitivity and specificity, offering new tools for calcium imaging in living cells. The findings highlight the potential of cpGFPs as powerful tools for studying calcium signaling in cellular processes.