RRC ID 49237
Author Nagai T, Sawano A, Park ES, Miyawaki A.
Title Circularly permuted green fluorescent proteins engineered to sense Ca2+.
Journal Proc Natl Acad Sci U S A
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.
Volume 98(6)
Pages 3197-202
Published 2001-3-13
DOI 10.1073/pnas.051636098
PII 051636098
PMID 11248055
PMC PMC30630
MeSH Calcium / metabolism* Calmodulin / genetics Calmodulin / metabolism Cations, Divalent Dimerization Gene Expression Genes, Reporter Genetic Engineering Genetic Variation Green Fluorescent Proteins HeLa Cells Humans Luminescent Proteins / genetics Luminescent Proteins / metabolism* Myosin-Light-Chain Kinase / genetics Myosin-Light-Chain Kinase / metabolism Nuclear Envelope / metabolism Nuclear Envelope / physiology Peptide Fragments / genetics Peptide Fragments / metabolism Permeability Recombinant Fusion Proteins / genetics Recombinant Fusion Proteins / metabolism
IF 9.58
Times Cited 662
WOS Category BIOCHEMISTRY & MOLECULAR BIOLOGY
Resource
DNA material Flash pericam/pcDNA3 (RDB15139) Inverse pericam/pcDNA3 (RDB15140) Ratiometric pericam/pcDNA3 (RDB15143) Ratiometric pericam-mt/pcDNA3 (RDB15144) Ratiometric pericam-nu/pcDNA3 (RDB15145) Split pericam-N/pcDNA3 (RDB15141) Split pericam-C/pcDNA3 (RDB15142).