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. Author manuscript; available in PMC: 2021 Feb 18.
Published in final edited form as: Biochemistry. 2020 Jan 21;59(6):802–817. doi: 10.1021/acs.biochem.9b01012

Figure 6. Co(II)-binding titrations indicate different metal-binding motifs for MRP126 variants.

Figure 6.

Optical absorption spectra for Co(II)-binding titrations (0–10 equivalents) of MRP126 and variants (240 μM) in 75 mM HEPES, 100 mM NaCl, pH 7.0, with 0.2 mM TCEP with (A-E) no Ca(II) added or (F-J) 6 mM Ca(II) added. (A,F) MRP126, (B,G) ΔTail, (C,H) ΔHis3Asp, (D,I) C5S, (E,J) C102S. Each line represents an addition of 150 μM Co(II) (0.625 equivalents). In each panel, the spectrum corresponding to 3.1 equivalents Co(II) is shown as a bolder line. The ε values were calculated assuming that all metal-binding sites occur in pairs in the MRP126 homodimer; these values therefore encompass at least two distinct Co(II)-protein interactions for most variants. All panels are representative of at least two independent experiments. See also Figure S16 for versions of these plots that include the 300–400 nm range.