Bioluminescent indicators for Ca2+ based on split Renilla luciferase complementation in living cells.
ABSTRACT Genetically encoded bioluminescent indicators for intracellular Ca2+ are described here with CaM-M13 interaction-induced complementation of split Renilla luciferase. The Ca2+-induced interaction between CaM and M13 leads to complementation of the N- and C-terminal halves of split Renilla luciferase in living cells. This intramolecular interaction results in the spontaneous and simultaneous emission of bioluminescence split Renilla luciferase. This is how intracellular Ca2+ is illuminated with the intramolecular complementation of split Renilla luciferase. The Ca2+-dependent spontaneous and simultaneous emission of bioluminescence promises to reveal Ca2+ dynamics in living cells, and also in vivo using the present indicators.
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ABSTRACT: The three-dimensional solution structure of the complex between calcium-bound calmodulin (Ca(2+)-CaM) and a 26-residue synthetic peptide comprising the CaM binding domain (residues 577 to 602) of skeletal muscle myosin light chain kinase, has been determined using multidimensional heteronuclear filtered and separated nuclear magnetic resonance spectroscopy. The two domains of CaM (residues 6 to 73 and 83 to 146) remain essentially unchanged upon complexation. The long central helix (residues 65 to 93), however, which connects the two domains in the crystal structure of Ca(2+)-CaM, is disrupted into two helices connected by a long flexible loop (residues 74 to 82), thereby enabling the two domains to clamp residues 3 to 21 of the bound peptide, which adopt a helical conformation. The overall structure of the complex is globular, approximating an ellipsoid of dimensions 47 by 32 by 30 angstroms. The helical peptide is located in a hydrophobic channel that passes through the center of the ellipsoid at an angle of approximately 45 degrees with its long axis. The complex is mainly stabilized by hydrophobic interactions which, from the CaM side, involve an unusually large number of methionines. Key residues of the peptide are Trp4 and Phe17, which serve to anchor the amino- and carboxyl-terminal halves of the peptide to the carboxyl- and amino-terminal domains of CaM, respectively. Sequence comparisons indicate that a number of peptides that bind CaM with high affinity share this common feature containing either aromatic residues or long-chain hydrophobic ones separated by a stretch of 12 residues, suggesting that they interact with CaM in a similar manner.Science 06/1992; 256(5057):632-8. · 31.03 Impact Factor
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ABSTRACT: THE protein aequorin, from the jellyfish Aequorea, emits bluish light in aqueous solution on addition specifically of calcium or strontium; the necessary energy of more than 60 kcalories/mole at the peak wavelength is evidently provided by an intramolecular reaction which is accompanied by a change in absorption spectrum and yields a blue fluorescent reaction product1,2. The extreme sensitivity to Ca2+, much more than to Sr2+, provides a basis for quick microdetermination of Ca2+ in biological fluids3 and has been used in detecting the relation of Ca2+ to contraction of single muscle fibres4,5 and to activity of mitochondria6. The chief properties of aequorin, including the molecular weight (m.w.) of 30,000 by sedimentation, diffusion or 32,000 by amino-acid composition, have been reported7. The kinetics of the light-emitting reaction have also been studied8.Nature 10/1970; 227(5265):1356-7. · 38.60 Impact Factor
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ABSTRACT: Fluorescence resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for various cellular functions. Although most indicators have cyan- and yellow-emitting fluorescent proteins (CFP and YFP) as FRET donor and acceptor, their poor dynamic range often prevents detection of subtle but significant signals. Here, we optimized the relative orientation of the two chromophores in the Ca(2+) indicator, yellow cameleon (YC), by fusing YFP at different angles. We generated circularly permuted YFPs (cpYFPs) that showed efficient maturation and acid stability. One of the cpYFPs incorporated in YC absorbs a great amount of excited energy from CFP in its Ca(2+)-saturated form, thereby increasing the Ca(2+)-dependent change in the ratio of YFP/CFP by nearly 600%. Both in cultured cells and in the nervous system of transgenic mice, the new YC enables visualization of subcellular Ca(2+) dynamics with better spatial and temporal resolution than before. Our study provides an important guide for the development and improvement of indicators using GFP-based FRET.Proceedings of the National Academy of Sciences 08/2004; 101(29):10554-9. · 9.74 Impact Factor