Circularly permuted green fluorescent proteins engineered to sense Ca2+

To visualize Ca2+-dependent protein-protein interactions in living cells by fluorescence readouts, we used a circularly permuted green fluorescent pro tein (cpGFP), in which the amino and carboxyl portions had been interchange d and reconnected by a short spacer between the original termini. The cpGFP was fused to carmodulin 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 Ca2+, probably because of the intera ction between calmodulin and M13 reading to an alteration of the environmen t surrounding the chromophore, Three types of pericam were obtained by muta ting several amino acids adjacent to the chromophore. Of these, "flash-peri cam" became brighter with Ca2+, whereas "inverse-pericam" dimmed. On the ot her hand, "ratiometric-pericam" had an excitation wavelength changing in a Ca2+-dependent manner. All of the pericams expressed in HeLa cells were abl e to monitor free Ca2+ dynamics, such as Ca2+ oscillations in the cytosol a nd the nucleus. Ca2+ imaging using high-speed confocal line-scanning micros copy and a flash-pericam allowed to detect the free propagation of Ca2+ ion s across the nuclear envelope. Then, free Ca2+ concentrations in the nucleu s and mitochondria were simultaneously measured by using ratiometric-perica ms having appropriate localization signals, revealing that extra-mitochondr ial Ca2+ transients caused rapid changes in the concentration of mitochondr ial Ca2+. Finally, a "split-pericam" was made by deleting the linker in the flash-pericam. The Ca2+-dependent interaction between calmodulin and M13 i n HeLa cells was monitored by the association of the two halves of GFP, nei ther of which was fluorescent by itself.