Biochemistry

Variations on a Theme

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Science  23 Mar 2001:
Vol. 291, Issue 5512, pp. 2279
DOI: 10.1126/science.291.5512.2279b

A change in fluorescence offers a sensitive signal that usually can be measured without compromising cellular integrity. Application of this approach to intact cells has enabled temporal and spatial observation of changes in ion concentrations and in macromolecular association (via fluorescent resonance energy transfer). An early indicator, the jellyfish protein aequorin, has largely been supplanted by small molecules, which have been refined to discriminate between Mg2+ and Ca2+, for instance; more recently, green fluorescent protein (GFP) has emerged as a tunable indicator with the advantage of genetically based targeting.

Nagai et al. have developed a new generation of circularly permuted GFP variants incorporating calmodulin. Among the constructs are flash-pericam, a single-wavelength indicator with a Kd of 0.7 μM for Ca2+; inverse-pericam, whose fluorescence decreases with Ca2+; and ratiometric-pericam, whose excitation wavelength is calcium-dependent. This last indicator could be targeted intracellularly either by tacking on a nuclear localization signal or by using the signal sequence from cytochrome c oxidase, a mitochondrial protein. — GJC

Proc. Natl. Acad. Sci. U.S.A.98, 3197 (2001).

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