Circularly Permuted Green Fluorescent Proteins Engineered to Sense Ca2

• Published in: Proceedings of the National Academy of Sciences - PNAS 2001-03, Vol.98 (6), p.3197-3202
• Main Authors: Nagai, Takeharu, Sawano, Asako, Park, Eun Sun, Miyawaki, Atsushi
• Format: Article
• Language: English
• Subjects: Absorption spectra; Amino acids; Biological Sciences; Calcium; Calcium - metabolism; Calmodulin - genetics; Calmodulin - metabolism; Cations, Divalent; Cellular biology; Chromophores; Complementary DNA; Cytosol; Dimerization; Fluorescence; Gene Expression; Genes, Reporter; Genetic Engineering; Genetic Variation; Green Fluorescent Proteins; HeLa Cells; Humans; Imaging; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Mitochondria; Myosin-Light-Chain Kinase - genetics; Myosin-Light-Chain Kinase - metabolism; Nuclear Envelope - metabolism; Nuclear Envelope - physiology; Peptide Fragments - genetics; Peptide Fragments - metabolism; Permeability; Proteins; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.051636098

To visualize Ca2+-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 Ca2+, 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 Ca2+, whereas "inverse-pericam" dimmed. On the other hand, "ratiometric-pericam" had an excitation wavelength changing in a Ca2+-dependent manner. All of the pericams expressed in HeLa cells were able to monitor free Ca2+ dynamics, such as Ca2+ oscillations in the cytosol and the nucleus. Ca2+ imaging using high-speed confocal line-scanning microscopy and a flash-pericam allowed to detect the free propagation of Ca2+ ions across the nuclear envelope. Then, free Ca2+ concentrations in the nucleus and mitochondria were simultaneously measured by using ratiometric-pericams having appropriate localization signals, revealing that extra-mitochondrial Ca2+ transients caused rapid changes in the concentration of mitochondrial Ca2+. Finally, a "split-pericam" was made by deleting the linker in the flash-pericam. The Ca2+-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.