Table 1 In vitro properties of YTnC compared to NTnC.

From: NTnC-like genetically encoded calcium indicator with a positive and enhanced response and fast kinetics

Properties

Proteins

YTnC

GCaMP6fsat

NTnCapo

apo

sat

Absorbance maxima (nm)

413

495 (405)

497

505

Emission maxima (nm)

514

516 (516)

516

518

Quantum yielda

0.012

0.19 (0.03)

0.51

0.71 ± 0.05

ε (mM−1 cm−1)

28 ± 2b

29 ± 3 (20 ± 2)c

73.5b

108 ± 6b

Brightness vs EGFP (%)

Purified proteind

1

17 (2)

114

232

HeLa cellse

ND

114 ± 23

(62 ± 3)

ND

188 ± 29

(202 ± 32)

ΔF/F (%)

0 mM Mg2+

1060 ± 37

2900 ± 97

100 ± 15

1 mM Mg2+

290 ± 23

2600 ± 190

100 ± 35

pKa

5.2 ± 0.1, 8.2 ± 0.1

6.3 ± 0.1

6.34 ± 0.01

6.09 ± 0.07

Kd (nM)f

0 mM Mg2+

223 ± 10 [n = 1.4 ± 0.1]

375 ± 8 [n = 2.01 ± 0.08]

84 ± 6 [n = 1.9 ± 0.1]

1 mM Mg2+

410 ± 19 [n = 1.7 ± 0.2]

492 ± 10 [n = 2.23 ± 0.09]

192 ± 40

Kdkin (nM)f,g

230 ± 200 [n = 1.4 ± 0.1]

450 ± 300 [2.4 ± 0.1]

94 ± 9 [n = 2.3 ± 0.1]

kon (s−1 × M−n)g

1.9 ± 0.9 × 109

3.5 × 1015

6 × 1015

kobs (s−1)h

5.3 ± 0.3; 1.12 ± 0.06

0.63 ± 0.01

5.8 ± 0.1; 0.08 ± 0.001

koff (s−1)i

0.96 ± 0.01

2.1 ± 0.1

0.8 ± 0.1; 0.05 ± 0.01j

t1/2off (s)

0.78

0.35

3

Maturation half-time (min)k

ND

16

ND

23

Photobleaching half-time (s)l

ND

11 ± 4

40 ± 8

  1. aQYs were determined at pH 7.20. EGFP (QY = 0.6031) and mTagBFP2 (QY = 0.6432) were used as reference standards for 495- and 405–413-nm absorbing states, respectively. bExtinction coefficient was determined by alkaline denaturation. cExtinction coefficient was estimated relative to YTnCapo with the same absorbance at 280 nm. dBrightness was calculated as a product of the quantum yield and extinction coefficient. eBrightness was normalized to that of the control mCherry protein, which was equimolar expressed in HeLa cells using GFP-P2A-mCherry construct, where GFP was EGFP, YTnCsat or NTnCapo proteins, respectively (Supplementary Fig. 4). EGFP had a brightness of 100 ± 16% (100 ± 20%). Values in the brackets correspond to the conditions in the presence of DMEM medium supplemented with 20 mM HEPES, pH 7.40, 10% FBS, Glutamine, 50 U/ml penicillin, and 50 U/ml streptomycin. fHill coefficient is shown in square brackets. gKdkin, Hill coefficients and kon values were obtained by fitting the observed association rates (Fig. 2a–c) at 100–1300 nM Ca2+ concentrations (for YTnC and GCaMP6f) or 100–300 nM Ca2+ concentrations (for NTnC) to the equation kobs = kon × [Ca2+]n + koff (Fig. 2d). Kdkin = (koff/kon)1/n. hkobs values are shown for the Ca2+ concentration of 300 nM. First and second values correspond to fast and slow exponents, respectively. i Refined koff values were determined from the dissociation kinetics records (Fig. 2f). jIn contrast to YTnC and GCaMP6f dissociation kinetics, NTnC kinetics do not agree with the two-state model. The NTnC dissociation curve was fitted to double exponential. koff values were estimated from double exponential decay with individual exponent contributions of 0.48:0.52. kEGFP had a maturation half-time of 14 min. lEGFP had a photobleaching half-time of 170 ± 20 s.
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