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. 2021 Mar 10;478(5):1085–1116. doi: 10.1042/BCJ20200838

Figure 2. Examples of ‘turn-off’ probes (Mf2, quin-2 and DP peptides) and their restricted application windows.

Figure 2.

(a–c) Determination of Zn(II)-binding stoichiometry and affinity of protein FrmRE64H in 10 mM Hepes, pH 7.0, 100 mM NaCl, 400 mM KCl: (a) solution spectra of apo- and Zn(II)-bound probe ligands quin-2 (blue traces) and Mf2 (red traces); (b) Mf2 probe response upon titration of Zn(II) ions into a solution containing Mf2 probe (10.1 µM) and FrmRE64H (18.1 µM monomer); (c) quin-2 probe response upon titration of Zn(II) ions into a solution containing quin-2 probe (13.4 µM) and FmRE64H (42.7 µM). Solid lines are curve fits to a model describing protein competition with Mf2 or quin-2 for 0.75 equivalents of Zn(II) per FrmRE64H monomer (i.e. three sites per tetramer, KZn1–3) generated using Dyanfit [60] (curves can also be modelled equivalently via equation 13b). Dashed lines are simulated curves with KZn1–3 10-fold tighter or weaker than the fitted values. Adapted from ref. [34] with data supplied by Dr. D. Osman and Prof. N.J. Robinson. (d–f) Determination of Cu(II) binding stoichiometry and affinity of protein CopC-H85F, with and without a hexa-His (6H) purification tag in 50 mM Mops pH 7.4: (d) quenching of fluorescence spectra of probe DP2 upon titration with Cu(II) ions; (e) DP2 probe response (plotted as normalised fluorescence, relative to apo-probe) upon titration of Cu(II) ions into a solution of DP2 only (4.0 µM, black); DP2 and CopC-H85F (4.0 µM each, blue); or DP2 and CopC-H85F-6H (4.0 µM each, red). (f) DP4 probe response upon titration of Cu(II) ions into a solution of DP4 only (4.0 µM, black); DP2 and CopC-H85F (4.0 µM each, blue); or DP2 and CopC-H85F-6H (4.0 µM each, red). Dashed black and blue lines in (e,f) are simple interpolation of data; solid red lines are curve fits via equation 13b to determine the weaker affinity Cu(II) site in CopC-H85F-6H (log KD = −9.4 in (e)) and the tight-affinity Cu(II) sites in CopC-H85F and CopC-H85F-6H (indistinguishable log KD = −14.6 in (f)). Adapted from ref. [35].