Characterization of a CorA Mg²⁺ transport channel from Methanococcus jannaschii using a Thermofluor-based stability assay

Figures & data

Figure 1.  Variation in Sypro Orange fluorescence with temperature. (A) Raw fluorescence data, comparing the effect of MjCorA-2 (black line) with a control (buffer; grey line). The reaction buffer was 180 mM NaCl, 22 mM citric acid, 33 mM HEPES, and 44 mM CHES pH8. (B) First derivative (dF/dT) of the data in (A). The arrow indicates the T_MP value.

Figure 2.  Optimization of protein and Sypro Dye concentrations. (A) Variation in the value of first derivative fluorescence values at four different concentrations of MjCorA-2. The final assay concentration of protein used are indicated. Arrows indicate T_MP values and signal strength is measured between the signal trough; t, and peak; p. (B) Variation in the value of first derivative fluorescence values at five different concentrations of Sypro Orange. The final assay concentration of MjCorA-1 used was 0.3 mg/ml in an assay volume of 50 µl. For both experiments, the reaction buffer used was 200 mM NaCl, 50 mM TrisHCl pH 8.

Figure 3.  Spider plot of the variation in T_MP of MjCorA-2 with pH and salt concentration. pH was varied using a three component buffer system containing 22 mM sodium citrate, 33 mM HEPES, and 44 mM CHES [28]. Salt concentration was varied as indicated. The radial axis shows temperature (°C) and the perimeter axis shows pH.

Table I.  Additive screen reagent concentrations and calculated T_MP. Changes in melting event peak temperatures (T_MP) for MjCorA-2 were calculated for each additive with respect to a no additive control. In some cases, a second unfolding event was observed which has been designated T_MP2. The values marked with an asterisk gave no signal, however there is evidence the unfolding event occurred at temperatures higher than the limits of the detector. A selection of additives has been repeated in triplicate and the error has been shown to 1 standard deviation from the mean. All additives, including the no additive water control, were prepared in a final buffer concentration of 50 mM TrisHCl, 200 mM NaCl pH8.

Additive	Final concentration	TMP (°C)	Δ TMP (°C)	TMP 2 (°C)
No additive	-	76.7 (±0.2)	0.0 (±0.2)
MgCl2	1 mM	80.9 (±0.23)	4.2 (±0.23)
MgAc	1 mM	82.6	5.9
Mg(NO3)2	1 mM	81.3	4.6
MgSO4	1 mM	81.1	4.4
MnCl2	1 mM	89.9 (±0.7)	13.2 (±0.7)
CoCl2*	1 mM	>95	>18.3
NiCl2	1 mM	90.0	13.3
CaCl2	1 mM	79.3	2.6
Cobalt (III) hexa-amine	1 mM	85.0 (±0.91)	8.3 (±0.91)
Li2SO4	1 mM	75.6	−1.1	92.5
LiCl	1 mM	76.6	−0.1
KCl	1 mM	76.6	−0.1
NaAc	1 mM	76.3	−0.4
Na citrate	1 mM	76.0	−0.7
AmSO4	1 mM	76.2	−0.5
AmCl2	1 mM	76.7	0.0
EDTA + MgCl2	2 mM, 1 mM	75.5	−1.2
EDTA	2 mM	75.9	−0.8
Imidazole	200 mM	76.4	−0.3
Imidazole	20 mM	76.9	0.2
His	20 mM	76.4	−0.3
His	2 mM	76.4	−0.3
All amino acids	2 mM	75.8 (±0.42)	−0.9 (±0.42)
Glu + Arg	50 mM	76.3	−0.4
Trp + Ile + Tyr + Leu + Phe	2 mM	76.3	−0.4
Spermidine	1 mM	78.8	2.1
-mercaptoethanol	0.05%	76.5	−0.2
DTT	1 mM	76.4	−0.3
CHAPS	0.01%	78.2	1.5
Nonyl Maltoside	0.02%	77.2	0.5
NV-10	0.25%	82.2 (±0.21)	5.5 (±0.21)
Glycerol	5%	79.0	2.3
Glycerol	20%	81.5	4.8
GuHCl	0.5 M	83.4	6.7
GuHCl	2 M	78.8	2.1	88.1
GuHCl	4 M	70.6	−6.1	86.3
GuHCl	6 M	47.8	−28.9
Urea	1 M	74.9	−1.8
Urea	4 M	71.9	−4.8
Urea + CoCl2	4 M, 1 mM	76.0	−0.7

Figure 4.  Comparison of T_MP for chymotrypsin-cleaved and un-cleaved MjCorA-2. pH was varied using a three component buffer system containing 22 mM sodium citrate, 33 mM HEPES, and 44 mM CHES [28] with 180 mM NaCl. Values marked by white squares gave readings off the measurable scale.

Supplementary Figure 1.  The effect of free detergent concentration. (A) Raw fluorescence data of chmotrypsin-cleaved MjCorA-2, comparing the effect of four different concentrations of reaction buffer detergent. The reaction buffer was 200 mM NaCl, 50 mM Tris-HCl pH8. (B) First derivative (dF/dT) of the data in (A).

Supplementary Figure 2.  The effect of experimental volume. (A) Variation in the value of first derivative fluorescence values of MjCorA-2 at three different experimental volumes. The reaction buffer was 200 mM NaCl, 50 mM Tris-HCl pH8.