Single fluorescent protein-based Ca2+ sensors with increased dynamic range

Ekaterina A. Souslova, Vsevolod V. Belousov, John G. Lock, Staffan Strömblad, Sergey Kasparov, Alexey P. Bolshakov, Vsevolod G. Pinelis, Yulii A. Labas, Sergey Lukyanov, Lorenz M. Mayr, Dmitriy M. Chudakov

Research output: Contribution to journalArticlepeer-review

92 Citations (Scopus)

Abstract

Background: Genetically encoded sensors developed on the basis of green fluorescent protein (GFP)-like proteins are becoming more and more popular instruments for monitoring cellular analytes and enzyme activities in living cells and transgenic organisms. In particular, a number of Ca2+ sensors have been developed, either based on FRET (Fluorescence Resonance Energy Transfer) changes between two GFP-mutants or on the change in fluorescence intensity of a single circularly permuted fluorescent protein (cpFP). Results: Here we report significant progress on the development of the latter type of Ca2+ sensors. Derived from the knowledge of previously reported cpFP-based sensors, we generated a set of cpFP-based indicators with different spectral properties and fluorescent responses to changes in Ca2+ concentration. Two variants, named Case12 and Case16, were characterized by particular high brightness and superior dynamic range, up to 12-fold and 16.5-fold increase in green fluorescence between Ca2+-free and Ca2+-saturated forms. We demonstrated the high potential of these sensors on various examples, including monitoring of Ca2+ response to a prolonged glutamate treatment in cortical neurons. Conclusion: We believe that expanded dynamic range, high brightness and relatively high pH-stability should make Case12 and Case16 popular research tools both in scientific studies and high throughput screening assays.

Original languageEnglish
Article number37
JournalBMC Biotechnology
Volume7
DOIs
Publication statusPublished - 29 Jun 2007
Externally publishedYes

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