title: Evaluation of the calmodulin-SOX9 interaction by "magnetic fishing" coupled to mass spectrometry.
abstract: Disruption 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.
authors.affiliation: Biointerfaces Institute and Department of Chemistry and Chemical Biology, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4M1 (Canada).
authors.forename: Meghan J
authors.initials: MJ
authors.lastname: McFadden
authors.forename: Todd
authors.initials: T
authors.lastname: Hryciw
authors.forename: Arthur
authors.initials: A
authors.lastname: Brown
authors.forename: Murray S
authors.initials: MS
authors.lastname: Junop
authors.forename: John D
authors.initials: JD
authors.lastname: Brennan
mesh_terms: D001665:Binding Sites; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D008280:Magnetics; D013058:Mass Spectrometry; D008555:Melitten / Q000737:chemistry / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D055758:SOX9 Transcription Factor / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D002147:Calmodulin; D055758:SOX9 Transcription Factor; D008555:Melitten
keywords: biological activity; fluorescence spectroscopy; inhibitors; mass spectrometry; protein-protein interactions