Evaluation of the Calmodulin‐SOX9 Interaction by “Magnetic Fishing” Coupled to Mass Spectrometry

AbstractDisruption of calmodulin (CaM)‐based protein interactions has been touted as a potential means for modulating several disease pathways. Among these is SOX9, which is a DNA binding protein that is involved in chrondrocyte differentiation and regulation of the hormones that control sexual development. In this work, we employed a “magnetic fishing”/mass spectrometry assay in conjunction with intrinsic fluorescence to examine the interaction of CaM with the CaM‐binding domain of SOX9 (SOX‐CAL), and to assess the modulation of this interaction by known anti‐CaM compounds. Our data show that there is a high affinity interaction between CaM and SOX‐CAL (27±9 nM), and that SOX‐CAL bound to the same location as the well‐known CaM antagonist melittin; unexpectedly, we also found that addition of CaM‐binding small molecules initially produced increased SOX‐CAL binding, indicative of binding to both the well‐known high‐affinity CaM binding site and a second, lower‐affinity binding site.

Evaluation of the Calmodulin‐SOX9 Interaction by “Magnetic Fishing” Coupled to Mass Spectrometry Journal Articles