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Table 1 In vitro properties of purified iYTnC2 compared to NTnC

From: Genetically encoded calcium indicator with NTnC-like design and enhanced fluorescence contrast and kinetics

Properties

Proteins

iYTnC2

NTnC

apo

sat

apo

sat

Absorbance maximum (nm)

499 (413)

410

505

Emission maximum (nm)

518 (516)

520

518

Quantum yielda

0.33 ± 0.03 (0.041 ± 0.005)

0.033 ± 0.004

0.71 ± 0.05

0.65 ± 0.04

ε (mM−1 cm− 1)

37 ± 1 (14.8 ± 0.4)c

31.6 ± 0.7b

108 ± 6b

52 ± 1c

Brightness (%)

16 (0.8)

1.4

100

44

Fluorescence contrast (fold)

0 mM Mg2+

8.1 ± 0.4

2.0 ± 0.3

1 mM Mg2+

5.5 ± 0.2

2.0 ± 0.7

pKa

7.4 ± 0.1

8.5 ± 0.1

6.09 ± 0.07

6.08 ± 0.02

Kd (nM)d

0 mM Mg2+

295 ± 9 [n = 1.7 ± 0.1]

84 ± 6 [n = 1.9 ± 0.1]

1 mM Mg2+

331 ± 22 [n = 1.6 ± 0.2]

192 ± 40 [n = 2.0 ± 0.4]

Kdkin (nM)e

200 ± 150 [n = 1.9 ± 0.1]

94 ± 9 [n = 2.3 ± 0.1]

kon (s−1× M-n)f

6.2 ± 0.5 × 1012

6 × 1015

koff (s−1)g

1.12 ± 0.01

0.8 ± 0.1; 0.05 ± 0.01h

t1/2off (s)g

0.58 ± 0.01

3.00 ± 0.05

Maturation half-time (min)i

3

ND

23

28

Photobleaching half-time (s)j

49 ± 19

ND

40 ± 8

70 ± 5

  1. aQYs were determined at pH 7.20. GCaMP6s in the saturated state (QY = 0.61 [18]) and mTagBFP2 (QY = 0.64 [19]) were used as reference standards for 499- to 505- and dim fluorescent 410- to 416- nm absorbing states, respectively
  2. bExtinction coefficients were determined by alkaline denaturation
  3. cExtinction coefficients were estimated relative to iYTnCsat or NTnCapo with the same concentration determined according to the absorbance at 280 nm
  4. dHill coefficients are shown in square brackets. In the absence and the presence of 1 mM Mg2+ ions, GCaMP6f control GECI had Kd value of 370 ± 8 [n = 2.03 ± 0.08] and 492 ± 10 [n = 2.23 ± 0.09] nM, respectively
  5. eKdkin, Hill coefficients and kon values were obtained via fitting the observed association rates to the equation kobs = kon × [Ca2+]n + koff (Fig. 2a). Kdkinetic = (koff/kon)1/n. Hill coefficients are shown in square brackets. GCaMP6f has Kdkin value of 450 ± 300 nM [n = 2.4 ± 0.1]
  6. fGCaMP6f has kon value of (3.5 ± 2) × 1015 s− 1× M-n
  7. gRefined koff and t1/2off values were determined from the dissociation kinetics records (Fig. 2d). GCaMP6f has t1/2off and koff values of 0.35 ± 0.02 s and 2.109 ± 0.002 s− 1, respectively
  8. hUnlike iYTnC kinetics, NTnC kinetics do not agree with the two-state model. NTnC kinetic curves were fitted to double exponentials. koff values were estimated from double exponential decay with individual exponent contributions of 0.48:0.52
  9. iEGFP had a maturation half-time of 14 min
  10. jmEGFP had a photobleaching half-time of 170 ± 20 s