Figure 2.

Folding and metal-binding characterizations of G4DFsc and 3His-G4DFsc. (a) UV-CD spectra of metal-free, Co(II)-bound, and Zn(II)-bound G4DFsc (top) and 3His-G4DFsc (bottom). Each sample contains 20 μM protein and 100 mM metal ions (where applicable). (b) Co(II) titration monitored by visible absorption spectroscopy for 100 μM G4DFsc (top) and 100 μM 3His-G4DFsc (bottom). The red lines are linear regressions of the initial and final linear regions of the curves to determine the stoichiometric ratio of Co(II) to protein. For these individual trials, the lines intersect at 225 μM for G4DFsc and 247 μM for 3His-G4DFsc, versus the theoretical value of 200 μM. The titrations have been performed in triplicate at slightly different protein concentrations, resulting in calculated Co(II):protein stoichiometries of 1.90 ±0.33 and 2.65±0.15. Insets display a representative Abs spectrum collected in the presence of 2 equivalents of Co(II) ions. Extinction coefficients at the peak maxima are 157 M⁻¹ cm⁻¹ for G4DFsc and 94 M⁻¹ cm⁻¹ for 3His-G4DFsc per Co(II) ion. (c) Fe(II) titration monitored by near-IR CD spectroscopy for G4DFsc (top) and 3His-G4DFsc (bottom). For both proteins, the titrations saturate at 2 equivalents of Fe(II).