Figure 2.

Titration of idoxifene with calmodulin. (a) The changes in fluorescence intensity of 10  $\mathrm{\mu }$ M idoxifene at 450 nm on addition of CaM up to 15  $\mathrm{\mu }$ M. Saturation is reached at a concentration ratio of CaM to idoxifene of 0.5, indicating the presence of two binding sites with sub-micromolar binding affinity. (b) Repeat of (a) but with 0.54  $\mathrm{\mu }$ M idoxifene and increasing CaM concentrations from 0.02  $\mathrm{\mu }$ M up to 0.6  $\mathrm{\mu }$ M. The data were fitted to a binding isotherm with a $\mathrm{2}:\mathrm{1}$ $\mathrm{idoxifene}:\mathrm{CaM}$ binding stoichiometry. (c) A region of the 1D ${}^{\mathrm{1}}$ H spectra acquired during the titration of idoxifene into 3 mM CaM. Solid lines highlight the completion of the slow exchange event for the assigned resonances at $\mathrm{2}:\mathrm{1}$ $\mathrm{idoxifene}:\mathrm{CaM}$ . Dotted lines are drawn for resonances undergoing shift changes beyond the end point of the titration. G134, I100, and I27 are representative of the slow exchange regime observed for CaM resonances during the titration, while N137 is representative of an intermediate exchange character.