Table 1.

Summary

		WT	Ser	Cys	Ala	Pro	Gly	Asn	Asp	Leu	Met	Arg	Trp
Expression (%)a		100	51.83 ± 2.3*	59.05 ± 7.3	56.41 ± 4.4	46.73 ± 5.4	57.89 ± 2.7	50.97 ± 9.6	45.89 ± 7.1	43.74 ± 7.0	47.76 ± 9.1	45.26 ± 4.2	44.69 ± 5.4
Fermentationb		Good	Poor	Good	S	S	S	Good	Good	Good	Good	No	No
Transport at 30 min (%)c	H+	100	47.8 ± 10.69	16.5 ± 2.01	34.0 ± 3.41	106.8 ± 4.54	18.5 ± 2.28	8.7 ± 2.83	8.0 ± 2.66	4.4 ± 1.42	49.6 ± 11.56	−1.0 ± 0.78	0.4 ± 0.12
	Na+	100	22.8 ± 0.18	23.0 ± 1.21	3.7 ± 0.25	11.3 ± 0.13	9.1 ± 1.10	2.5 ± 0.28	2.7 ± 0.12	1.0 ± 0.11	30.5 ± 4.02	0.5 ± 0.27	0.2 ± 0.12
	Li+	100	84.7 ± 1.18	75.3 ± 0.55	93.6 ± 5.48	109.2 ± 2.39	41.7 ± 4.15	22.5 ± 0.76	21.9 ± 0.83	15.0 ± 1.86	31.0 ± 5.09	−0.5 ± 0.39	0 ± 0.05
Sugar binding (IC50, mM)d	H+	+	+	+	+	+	+	+	+	+	+	+	−
	Na+	+	+	+	+	+	+	+	+	+	+	+	−
	Li+	0.73 ± 0.04	1.99 ± 0.12	2.58 ± 0.03	2.48 ± 0.04	5.77 ± 0.52	+	+	+	+	+	+	−
Na+ binding (K0.5(Na+), mM)e		0.83 ± 0.25	8.24 ± 0.22	30.34 ± 1.81	/**	/	+	+	−	−	+	−	−
Li+ binding (K0.5(Na+), mM)f		1.88 ± 0.35	3.48 ± 0.43	0.67 ± 0.01	0.58 ± 0.05	0.42 ± 0.09	+	+	+	+	+	−	−
Soluble MelBSt at 52 °C (%)g	Na+	41.6 ± 3.5	10.85 ± 4.0	3.89 ± 0.2	3.99 ± 0.5	3.32 ± 1.6	2.14 ± 2.1	3.50 ± 1.2	7.94 ± 1.7	1.38 ± 0.3	1.84 ± 1.2	2.87 ± 1.6	2.35 ± 0.1
	Li+	44.89 ± 4.0	33.78 ± 1.3	46.62 ± 1.3	51.51 ± 4.2	7.99 ± 2.2	8.68 ± 1.5	21.25 ± 3.0	3.79 ± 1.2	19.62 ± 0.5	31.51 ± 3.2	2.55 ± 0.9	2.73 ± 1.5
Tm (°C)h	Li+	62.54 ± 0.32		60.30 ± 0.18	64.71 ± 0.23
	Li++Mel	63.87 ± 0.04		61.03 ± 0.13
pKa estimationi	Mel	8.75 ± 0.02***	7.75 ± 0.02	8.36 ± 0.03	8.75 ± 0.02	9.18 ± 0.02

^aRelative expression derived from Fig. 3.

^bInterpretation of fermentation results presented in Fig. 4; S, Colonies were small but red.

^crelative transport activity at 30-min time point presented in Fig. 4.

^dall data were from Trp→D²G assays. IC₅₀ were carried out with MelB_St in nanodiscs and the presence of Li⁺ (Fig. 7). The bound D²G was displaced by melibiose binding as described in the Methods. For most cases, the FRET binding results in Fig. 5 were explained as positive (+) or negative (−) qualitatively.

^eK_0.5(Na+), Na⁺ stimulation constant on the Trp→D²G FRET carried out with MelB_St in nanodiscs as determined in Fig. 6. For Ala and Pro mutants, no fitting due to lack of feature of the titration curve. For other cases, the Na⁺ binding data in Fig. 5 were explained as positive (+) or negative (−) qualitatively.

^fK_0.5(Li+), Li⁺ stimulation constant on the Trp→D²G FRET were carried out with MelB_St in nanodiscs as determined in Fig. 6. For Ala and Pro mutants, no fitting due to lack of feature of the titration curve. For other cases, the Li⁺ binding data in Fig. 5 were explained as positive (+) or negative (−) qualitatively.

^gData were from Fig. 8, and the relative amount of soluble MelB_St proteins compared to the total MelB_St after 52 °C treatment as described in Methods.

^hthe melting temperature was determined by CD spectroscopy in Fig. 9. All purified protein samples contain 0.01% DDM and 100 mM LiCl in the assay buffer.

ⁱthe calculated pKa values by PROPKA program as described in Methods.

^*SE, number of tests = 2-3

^**No fitting.

^***SE, number of tests = 1000.