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Generating a Metal-responsive Transcriptional Regulator to Test What Confers Metal Sensing in Cells.

Osman D, Piergentili C, Chen J, Chakrabarti B, Foster AW, Lurie-Luke E, Huggins TG, Robinson NJ - J. Biol. Chem. (2015)

Bottom Line: Unexpectedly, FrmR was found to already bind Co(II), Zn(II), and Cu(I), and moreover metals, as well as formaldehyde, trigger an allosteric response that weakens DNA affinity.Counter-intuitively, the allosteric coupling free energy for Zn(II) is smaller in metal-sensing FrmRE64H compared with nonsensing FrmR.By determining the copies of FrmR and FrmRE64H tetramers per cell, then estimating promoter occupancy as a function of intracellular Zn(II) concentration, we show how a modest tightening of Zn(II) affinity, plus weakened DNA affinity of the apoprotein, conspires to make the relative properties of FrmRE64H (compared with ZntR and Zur) sufficient to sense Zn(II) inside cells.

View Article: PubMed Central - PubMed

Affiliation: From the School of Biological and Biomedical Sciences and Department of Chemistry, Durham University, Durham DH1 3LE, United Kingdom.

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Zn(II), Co(II), and Cu(I) affinities of FrmRE64H and FrmR.A, representative (n = 3) mag fura-2 absorbance upon titration of mag fura-2 (10.1 μm) with ZnCl2 in the presence of FrmRE64H (18.8 μm, monomer). B, as A but with mag fura-2 (12.2 μm) and FrmR (20.4 μm, monomer). C, representative (n = 3) quin-2 absorbance upon titration of quin-2 (13.4 μm) with ZnCl2 in the presence of FrmRE64H (42.7 μm, monomer). D, as C but with quin-2 (14.1 μm) and FrmR (39.9 μm, monomer). In each case (A–D), solid lines are fits to a model describing protein competition with mag fura-2 or quin-2 for 0.75 equivalents of Zn(II) per monomer (three sites per tetramer, KZn1–3). Dashed lines are simulated curves with KZn1–3 10-fold tighter and 10-fold weaker. E, representative (n = 3) fura-2 fluorescence emission (λex = 360 nm) upon titration of fura-2 (9.8 μm) with CoCl2 in the presence (filled circles) of FrmRE64H (49.3 μm, monomer). Solid line is a fit to a model describing protein competition for 0.25 eq of Co(II) per monomer (one site per tetramer, KCo1). Dashed lines are simulated curves describing KCo1 10-fold tighter and 10-fold weaker. F, fluorescence emission of fura-2 (10.3 μm) upon titration with Co(II) as described in E in the absence (open circles) or presence (filled circles) of FrmR (41.9 μm, monomer). G, representative (n = 3) Co(II)-dependent absorbance at 336 nm of FrmRE64H (87.0 μm, monomer) upon titration with CoCl2 in the presence of 50 mm BisTris. H, as G but with FrmR (83.9 μm, monomer). Solid lines (for G and H), are fits to a model describing protein competition for 1 m eq of Co(II) per monomer (four sites per tetramer, KCo1–4). Dashed lines are simulated curves describing KCo1–4 10-fold tighter and 10-fold weaker. I, representative (n = 3) BCA absorbance upon titration of BCA (40 μm) with CuCl in the presence of FrmRE64H (11 μm, monomer). J, as I but with FrmR (10 μm, monomer). Solid lines (for I and J) are fits to a model describing protein competition with BCA for 2 eq of Cu(I) per monomer (eight sites per tetramer). Dashed lines are simulated curves with KCu1–2 10-fold tighter and 10-fold weaker. Solid red line (I only) is a simulated curve describing KCu1–2 100-fold weaker. K, representative (n = 4) BCS absorbance upon titration of BCS (10 μm) with CuCl in the presence of FrmRE64H (29.7 μm, monomer).
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Figure 4: Zn(II), Co(II), and Cu(I) affinities of FrmRE64H and FrmR.A, representative (n = 3) mag fura-2 absorbance upon titration of mag fura-2 (10.1 μm) with ZnCl2 in the presence of FrmRE64H (18.8 μm, monomer). B, as A but with mag fura-2 (12.2 μm) and FrmR (20.4 μm, monomer). C, representative (n = 3) quin-2 absorbance upon titration of quin-2 (13.4 μm) with ZnCl2 in the presence of FrmRE64H (42.7 μm, monomer). D, as C but with quin-2 (14.1 μm) and FrmR (39.9 μm, monomer). In each case (A–D), solid lines are fits to a model describing protein competition with mag fura-2 or quin-2 for 0.75 equivalents of Zn(II) per monomer (three sites per tetramer, KZn1–3). Dashed lines are simulated curves with KZn1–3 10-fold tighter and 10-fold weaker. E, representative (n = 3) fura-2 fluorescence emission (λex = 360 nm) upon titration of fura-2 (9.8 μm) with CoCl2 in the presence (filled circles) of FrmRE64H (49.3 μm, monomer). Solid line is a fit to a model describing protein competition for 0.25 eq of Co(II) per monomer (one site per tetramer, KCo1). Dashed lines are simulated curves describing KCo1 10-fold tighter and 10-fold weaker. F, fluorescence emission of fura-2 (10.3 μm) upon titration with Co(II) as described in E in the absence (open circles) or presence (filled circles) of FrmR (41.9 μm, monomer). G, representative (n = 3) Co(II)-dependent absorbance at 336 nm of FrmRE64H (87.0 μm, monomer) upon titration with CoCl2 in the presence of 50 mm BisTris. H, as G but with FrmR (83.9 μm, monomer). Solid lines (for G and H), are fits to a model describing protein competition for 1 m eq of Co(II) per monomer (four sites per tetramer, KCo1–4). Dashed lines are simulated curves describing KCo1–4 10-fold tighter and 10-fold weaker. I, representative (n = 3) BCA absorbance upon titration of BCA (40 μm) with CuCl in the presence of FrmRE64H (11 μm, monomer). J, as I but with FrmR (10 μm, monomer). Solid lines (for I and J) are fits to a model describing protein competition with BCA for 2 eq of Cu(I) per monomer (eight sites per tetramer). Dashed lines are simulated curves with KCu1–2 10-fold tighter and 10-fold weaker. Solid red line (I only) is a simulated curve describing KCu1–2 100-fold weaker. K, representative (n = 4) BCS absorbance upon titration of BCS (10 μm) with CuCl in the presence of FrmRE64H (29.7 μm, monomer).

Mentions: As the FrmRE64H variant, but not FrmR, responds to Zn(II) and cobalt in cells, it was anticipated that this substitution had succeeded in tightening the affinity for these metals. The chromophores mag fura-2 and quin-2 form 1:1 complexes with Zn(II) and undergo concomitant changes in absorbance upon metal binding, which can be used to monitor competition with proteins and hence to estimate protein KZn(II) (11, 13, 44–48, 51). Titration of 10.1 μm or 12.2 μm mag fura-2 with Zn(II) in the presence of FrmRE64H (18.8 μm, monomer) or FrmR (20.4 μm, monomer), respectively, gave negligible change in absorbance up to 0.5–0.75 eq of Zn(II) per protein monomer, implying competition with the chromophore for metal (Fig. 4, A and B). At these protein concentrations, CsoR/RcnR family members exist as tetramers with four metal-binding sites per tetramer, and with some evidence of negative cooperativity between sites (12, 17, 18, 52). A 1:1 stoichiometry equating to four Zn(II) per tetramer was observed for both FrmRE64H and FrmR (Fig. 3, D and H), but the fourth sites are too weak to compete with mag fura-2 (hence competition is complete after addition of ∼24.2 and ∼27.5 μm Zn(II) (Fig. 4, A and B)). Data were fit to models describing tight binding of 3 m eq of Zn(II)/tetramer, with dashed lines representing simulated curves describing KZn1–3 10-fold tighter or 10-fold weaker than the calculated affinity (Fig. 4, A and B). For both proteins, this suggests KZn1–3 at or approaching the tighter limit of the assay using mag fura-2 (KZn(II)mag fura-2 = 2.0 × 10−8m). Competitions were therefore conducted with 13.4 or 14.1 μm quin-2 (KZn(II)quin-2 = 3.7 × 10−12m) and FrmRE64H (42.7 μm, monomer) or FrmR (39.9 μm, monomer), respectively (Fig. 4, C and D). Again, data were fit to models describing binding of 3 m eq of Zn(II)/tetramer (as expected, the fourth sites did not show competition with quin-2) with dashed lines in Fig. 4, C and D, describing simulated curves for KZn1–3 10-fold tighter or 10-fold weaker than the calculated affinity of the proteins. Mean values of KZn1–3 2.33 (±0.3) × 10−11m and 1.7 (±0.7) × 10−10m for FrmRE64H and FrmR, respectively, are thus within the range of this assay (Fig. 4, C and D, and Table 1).


Generating a Metal-responsive Transcriptional Regulator to Test What Confers Metal Sensing in Cells.

Osman D, Piergentili C, Chen J, Chakrabarti B, Foster AW, Lurie-Luke E, Huggins TG, Robinson NJ - J. Biol. Chem. (2015)

Zn(II), Co(II), and Cu(I) affinities of FrmRE64H and FrmR.A, representative (n = 3) mag fura-2 absorbance upon titration of mag fura-2 (10.1 μm) with ZnCl2 in the presence of FrmRE64H (18.8 μm, monomer). B, as A but with mag fura-2 (12.2 μm) and FrmR (20.4 μm, monomer). C, representative (n = 3) quin-2 absorbance upon titration of quin-2 (13.4 μm) with ZnCl2 in the presence of FrmRE64H (42.7 μm, monomer). D, as C but with quin-2 (14.1 μm) and FrmR (39.9 μm, monomer). In each case (A–D), solid lines are fits to a model describing protein competition with mag fura-2 or quin-2 for 0.75 equivalents of Zn(II) per monomer (three sites per tetramer, KZn1–3). Dashed lines are simulated curves with KZn1–3 10-fold tighter and 10-fold weaker. E, representative (n = 3) fura-2 fluorescence emission (λex = 360 nm) upon titration of fura-2 (9.8 μm) with CoCl2 in the presence (filled circles) of FrmRE64H (49.3 μm, monomer). Solid line is a fit to a model describing protein competition for 0.25 eq of Co(II) per monomer (one site per tetramer, KCo1). Dashed lines are simulated curves describing KCo1 10-fold tighter and 10-fold weaker. F, fluorescence emission of fura-2 (10.3 μm) upon titration with Co(II) as described in E in the absence (open circles) or presence (filled circles) of FrmR (41.9 μm, monomer). G, representative (n = 3) Co(II)-dependent absorbance at 336 nm of FrmRE64H (87.0 μm, monomer) upon titration with CoCl2 in the presence of 50 mm BisTris. H, as G but with FrmR (83.9 μm, monomer). Solid lines (for G and H), are fits to a model describing protein competition for 1 m eq of Co(II) per monomer (four sites per tetramer, KCo1–4). Dashed lines are simulated curves describing KCo1–4 10-fold tighter and 10-fold weaker. I, representative (n = 3) BCA absorbance upon titration of BCA (40 μm) with CuCl in the presence of FrmRE64H (11 μm, monomer). J, as I but with FrmR (10 μm, monomer). Solid lines (for I and J) are fits to a model describing protein competition with BCA for 2 eq of Cu(I) per monomer (eight sites per tetramer). Dashed lines are simulated curves with KCu1–2 10-fold tighter and 10-fold weaker. Solid red line (I only) is a simulated curve describing KCu1–2 100-fold weaker. K, representative (n = 4) BCS absorbance upon titration of BCS (10 μm) with CuCl in the presence of FrmRE64H (29.7 μm, monomer).
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Figure 4: Zn(II), Co(II), and Cu(I) affinities of FrmRE64H and FrmR.A, representative (n = 3) mag fura-2 absorbance upon titration of mag fura-2 (10.1 μm) with ZnCl2 in the presence of FrmRE64H (18.8 μm, monomer). B, as A but with mag fura-2 (12.2 μm) and FrmR (20.4 μm, monomer). C, representative (n = 3) quin-2 absorbance upon titration of quin-2 (13.4 μm) with ZnCl2 in the presence of FrmRE64H (42.7 μm, monomer). D, as C but with quin-2 (14.1 μm) and FrmR (39.9 μm, monomer). In each case (A–D), solid lines are fits to a model describing protein competition with mag fura-2 or quin-2 for 0.75 equivalents of Zn(II) per monomer (three sites per tetramer, KZn1–3). Dashed lines are simulated curves with KZn1–3 10-fold tighter and 10-fold weaker. E, representative (n = 3) fura-2 fluorescence emission (λex = 360 nm) upon titration of fura-2 (9.8 μm) with CoCl2 in the presence (filled circles) of FrmRE64H (49.3 μm, monomer). Solid line is a fit to a model describing protein competition for 0.25 eq of Co(II) per monomer (one site per tetramer, KCo1). Dashed lines are simulated curves describing KCo1 10-fold tighter and 10-fold weaker. F, fluorescence emission of fura-2 (10.3 μm) upon titration with Co(II) as described in E in the absence (open circles) or presence (filled circles) of FrmR (41.9 μm, monomer). G, representative (n = 3) Co(II)-dependent absorbance at 336 nm of FrmRE64H (87.0 μm, monomer) upon titration with CoCl2 in the presence of 50 mm BisTris. H, as G but with FrmR (83.9 μm, monomer). Solid lines (for G and H), are fits to a model describing protein competition for 1 m eq of Co(II) per monomer (four sites per tetramer, KCo1–4). Dashed lines are simulated curves describing KCo1–4 10-fold tighter and 10-fold weaker. I, representative (n = 3) BCA absorbance upon titration of BCA (40 μm) with CuCl in the presence of FrmRE64H (11 μm, monomer). J, as I but with FrmR (10 μm, monomer). Solid lines (for I and J) are fits to a model describing protein competition with BCA for 2 eq of Cu(I) per monomer (eight sites per tetramer). Dashed lines are simulated curves with KCu1–2 10-fold tighter and 10-fold weaker. Solid red line (I only) is a simulated curve describing KCu1–2 100-fold weaker. K, representative (n = 4) BCS absorbance upon titration of BCS (10 μm) with CuCl in the presence of FrmRE64H (29.7 μm, monomer).
Mentions: As the FrmRE64H variant, but not FrmR, responds to Zn(II) and cobalt in cells, it was anticipated that this substitution had succeeded in tightening the affinity for these metals. The chromophores mag fura-2 and quin-2 form 1:1 complexes with Zn(II) and undergo concomitant changes in absorbance upon metal binding, which can be used to monitor competition with proteins and hence to estimate protein KZn(II) (11, 13, 44–48, 51). Titration of 10.1 μm or 12.2 μm mag fura-2 with Zn(II) in the presence of FrmRE64H (18.8 μm, monomer) or FrmR (20.4 μm, monomer), respectively, gave negligible change in absorbance up to 0.5–0.75 eq of Zn(II) per protein monomer, implying competition with the chromophore for metal (Fig. 4, A and B). At these protein concentrations, CsoR/RcnR family members exist as tetramers with four metal-binding sites per tetramer, and with some evidence of negative cooperativity between sites (12, 17, 18, 52). A 1:1 stoichiometry equating to four Zn(II) per tetramer was observed for both FrmRE64H and FrmR (Fig. 3, D and H), but the fourth sites are too weak to compete with mag fura-2 (hence competition is complete after addition of ∼24.2 and ∼27.5 μm Zn(II) (Fig. 4, A and B)). Data were fit to models describing tight binding of 3 m eq of Zn(II)/tetramer, with dashed lines representing simulated curves describing KZn1–3 10-fold tighter or 10-fold weaker than the calculated affinity (Fig. 4, A and B). For both proteins, this suggests KZn1–3 at or approaching the tighter limit of the assay using mag fura-2 (KZn(II)mag fura-2 = 2.0 × 10−8m). Competitions were therefore conducted with 13.4 or 14.1 μm quin-2 (KZn(II)quin-2 = 3.7 × 10−12m) and FrmRE64H (42.7 μm, monomer) or FrmR (39.9 μm, monomer), respectively (Fig. 4, C and D). Again, data were fit to models describing binding of 3 m eq of Zn(II)/tetramer (as expected, the fourth sites did not show competition with quin-2) with dashed lines in Fig. 4, C and D, describing simulated curves for KZn1–3 10-fold tighter or 10-fold weaker than the calculated affinity of the proteins. Mean values of KZn1–3 2.33 (±0.3) × 10−11m and 1.7 (±0.7) × 10−10m for FrmRE64H and FrmR, respectively, are thus within the range of this assay (Fig. 4, C and D, and Table 1).

Bottom Line: Unexpectedly, FrmR was found to already bind Co(II), Zn(II), and Cu(I), and moreover metals, as well as formaldehyde, trigger an allosteric response that weakens DNA affinity.Counter-intuitively, the allosteric coupling free energy for Zn(II) is smaller in metal-sensing FrmRE64H compared with nonsensing FrmR.By determining the copies of FrmR and FrmRE64H tetramers per cell, then estimating promoter occupancy as a function of intracellular Zn(II) concentration, we show how a modest tightening of Zn(II) affinity, plus weakened DNA affinity of the apoprotein, conspires to make the relative properties of FrmRE64H (compared with ZntR and Zur) sufficient to sense Zn(II) inside cells.

View Article: PubMed Central - PubMed

Affiliation: From the School of Biological and Biomedical Sciences and Department of Chemistry, Durham University, Durham DH1 3LE, United Kingdom.

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Related in: MedlinePlus