Volume 451 (2013), pp. 365–374
Figures 3, 5, 6 and 7 should have been published in colour. Colour versions of these Figures and corrected caption to Figure 7 appear here.
The involvement of the polar residues in Co2+ transport
The growth activity of TmCorA with the Asn288, Thr295 and Thr299 mutated to either (A) leucine or (B) methionine was monitored in the presence of various Co2+ concentrations. A reduction in growth activity upon Co2+ concentration increase is indicative of the Co2+ transport activity of the TmCorA variant. The wild-type TmCorA (WT corA) and the empty CorA-less pBAD vector (corA-less) were used as positive and negative controls respectively. The results are the mean±S.D. of at least three independent experiments.
The growth activity of TmCorA with the Asn288, Thr295 and Thr299 mutated to either (A) leucine or (B) methionine was monitored in the presence of various Co2+ concentrations. A reduction in growth activity upon Co2+ concentration increase is indicative of the Co2+ transport activity of the TmCorA variant. The wild-type TmCorA (WT corA) and the empty CorA-less pBAD vector (corA-less) were used as positive and negative controls respectively. The results are the mean±S.D. of at least three independent experiments.
Exploring the Co2+ transport ability of threonine residues in the channel
The growth activity of TmCorA with the T295S, T299S or T305S mutant was monitored in the presence of various Co2+ concentrations. A reduction in growth activity upon Co2+ concentration increase is indicative of the Co2+ transport activity of the TmCorA variant. The wild-type TmCorA (WT corA) and the empty CorA-less pBAD vector (corA-less) were used as positive and negative controls respectively. The results are the means±S.D. of at least three independent experiments.
The growth activity of TmCorA with the T295S, T299S or T305S mutant was monitored in the presence of various Co2+ concentrations. A reduction in growth activity upon Co2+ concentration increase is indicative of the Co2+ transport activity of the TmCorA variant. The wild-type TmCorA (WT corA) and the empty CorA-less pBAD vector (corA-less) were used as positive and negative controls respectively. The results are the means±S.D. of at least three independent experiments.
Assaying the sensitivity of the T295S mutant towards Mg2+
The growth activity of the T295S mutant was monitored in the presence of increasing Co2+ concentrations, as well as a fixed Mg2+ concentration of (A) 0 μM, (B) 5 μM, (C) 25 μM and (D) 250 μM. A reduction in growth activity upon Co2+ concentration increase is indicative of the Co2+ transport activity of the TmCorA variant. The wild-type TmCorA (WT corA) and the empty CorA-less pBAD vector (corA-less) were used as positive and negative controls respectively. The results are the means±S.D. of at least three independent experiments.
The growth activity of the T295S mutant was monitored in the presence of increasing Co2+ concentrations, as well as a fixed Mg2+ concentration of (A) 0 μM, (B) 5 μM, (C) 25 μM and (D) 250 μM. A reduction in growth activity upon Co2+ concentration increase is indicative of the Co2+ transport activity of the TmCorA variant. The wild-type TmCorA (WT corA) and the empty CorA-less pBAD vector (corA-less) were used as positive and negative controls respectively. The results are the means±S.D. of at least three independent experiments.
The involvement of the M1-site in the stability and activity of TmCorA
(A) The structure of the M1-site with a Co2+ (green sphere) and the co-ordinating side chains. (B) The growth activity of TmCorA with the M1-site specific mutants was monitored in the presence of various Co2+ concentrations. A reduction in growth activity upon Co2+ concentration increase (Co2+ toxicity) is indicative of the Co2+ transport activity of the TmCorA variant. The wild-type TmCorA (WT corA) and the empty CorA-less pBAD vector (corA-less) were used as positive and negative controls respectively. The results are the means±S.D. of at least three independent experiments. (C) Thermostability of TmCorA wild-type and the M-site mutants in the presence of various Co2+ concentrations. Each sample was heated at 85°C for 10 min and the presence of stable protein was analysed by SDS/PAGE followed by Coomassie Blue staining. Untreated TmCorA was used as a positive control. The protein size was verified using Seeblue® Plus2 Pre-stained Standard (Invitrogen) (L). The concentration of Co2+ used is indicated from 0 to 1000 μM.
(A) The structure of the M1-site with a Co2+ (green sphere) and the co-ordinating side chains. (B) The growth activity of TmCorA with the M1-site specific mutants was monitored in the presence of various Co2+ concentrations. A reduction in growth activity upon Co2+ concentration increase (Co2+ toxicity) is indicative of the Co2+ transport activity of the TmCorA variant. The wild-type TmCorA (WT corA) and the empty CorA-less pBAD vector (corA-less) were used as positive and negative controls respectively. The results are the means±S.D. of at least three independent experiments. (C) Thermostability of TmCorA wild-type and the M-site mutants in the presence of various Co2+ concentrations. Each sample was heated at 85°C for 10 min and the presence of stable protein was analysed by SDS/PAGE followed by Coomassie Blue staining. Untreated TmCorA was used as a positive control. The protein size was verified using Seeblue® Plus2 Pre-stained Standard (Invitrogen) (L). The concentration of Co2+ used is indicated from 0 to 1000 μM.
