Figure 8.

Intrinsic fluorescence of EncFtn WT and variants titrated with Zn(II). Fluorescence emission of EncFtn proteins (20 µm, corresponding to [entry site] = 10 µm) at various wavelength (330 nm for EncFtn-WT and -E31A, 355 nm for EncFtn-E34A and E31A/E34A, and 322 nm for EncFtn-W38A), with excitation at 280 nm, and following titration with ZnSO₄.7H₂O. A and B, fluorescence is quenched upon zinc addition, with inflection around 1 molar equivalents, suggesting perturbation of environment around Trp-38. C and D, in the absence of entry-site residues available for metal coordination, no quenching was observed. E, EncFtn-W38A was tested as the control. Symbols represent average and standard deviation (error bars) of experimental replicates, collected at equilibrium either by Scan (n >3) or Kinetic (n = 10) options on the Cary Eclipse software package. Data were normalized to allow comparison. Dotted gray lines were added at 1 molar equivalent of Zn(II) when inflection was observed.