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


CONCLUSIONS
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.
Souslova
Ekaterina A
EA
Shemiakin-Ovchinnikov Institute of Bioorganic Chemistry, RAS, Miklukho-Maklaya 16/10, Moscow, Russia. souslova@gmail.com <souslova@gmail.com>
Belousov
Vsevolod V
VV
Lock
John G
JG
Strömblad
Staffan
S
Kasparov
Sergey
S
Bolshakov
Alexey P
AP
Pinelis
Vsevolod G
VG
Labas
Yulii A
YA
Lukyanov
Sergey
S
Mayr
Lorenz M
LM
Chudakov
Dmitriy M
DM
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D016716:PC12 Cells; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D002118:Calcium