Extended Data Fig. 5: Spectrofluorometric titrations of RE-LanM (Hans-LanM, Hans-LanM(R100K), and Mex-LanM) complexes with citrate as a competitor, monitored by intrinsic protein fluorescence.
From: Enhanced rare-earth separation with a metal-sensitive lanmodulin dimer
Emission values are normalized to 1.0 for the fluorescence of the apoprotein. Note that the fluorescence intensity of Hans-LanM’s Trp residues decreases going from the RE-bound to apo state (Supplementary Fig. 30), whereas the intensity of Mex’s Tyr residue increases going from the RE-bound to apo state4,6. Initial conditions: 20 μM protein, 40 μM RE, 20 mM acetate, 100 mM KCl, pH 5.0, for all experiments, into which increasing concentrations of citrate were titrated. The citrate concentrations at which 50% of each metal is desorbed under these conditions ([citrate]1/2) are summarized in Supplementary Table 11 and plotted in Fig. 4a. a, Hans-LanM. b, Hans-LanM(R100K). The compressed difference between the [citrate]1/2 values for La and Nd in Hans-LanM(R100K) vs. wild-type Hans-LanM illustrates the role of dimerization in enhancing affinity differences for the LREs, especially LaIII. c, Mex-LanM. Nd and Dy data were reported in Dong et al6. d, Comparison of the ratios of [citrate]1/2 value for Nd to that for Dy, for each protein, illustrating the greater Nd/Dy selectivity of Hans-LanM relative to Mex-LanM. The ratios for wild-type Hans-LanM and the R100K variant are not significantly different (p > 0.05) by two-tailed t-test, suggesting that the hydrogen-bonding network involving Arg100 contributes relatively little to NdIII/HRE selectivity, though it does impact LaIII selectivity significantly (Fig. 4a). All data are shown as mean ± s.d. (a—c) or s.e.m. (d) for data from 3 independent experiments.
