title: Human multiprotein bridging factor 1 and Calmodulin do not interact in vitro as confirmed by NMR spectroscopy and CaM-agarose affinity chromatography.
abstract: The human multiprotein bridging factor 1 (hMBF1) has been established in different cellular types to have the role of transcriptional coactivator. It is also reported to be a putative Calmodulin (CaM) target, able to bind CaM in its calcium-free state, but little is known about the structural features and the biological relevance of this interaction. We applied NMR to investigate the interaction between the two proteins in solution and compared the results with those obtained with CaM-agarose affinity chromatography. No changes in ¹H-¹⁵N HSQC spectrum of both apo-CaM and Ca²⁺-CaM upon addition of hMBF1 prove that the two proteins do not interact in vitro. These results were confirmed by CaM-agarose affinity chromatography when operating under the same conditions. The discrepancy between present and previous experiments performed with CaM-agarose affinity chromatography depends on different experimental parameters suggesting that particular attention must be paid when CaM, or other immobilized proteins, are used to measure their affinity with putative partners. These results also imply that if an interaction between the two proteins exists in vivo, as reported for hMBF1 of endothelial cells, it might involve a posttranslational modified form of the proteins or it relies on other conditions imposed by the cellular environment.
authors.affiliation: Department of Food Science, University of Bologna, Piazza Goidanich 60, 47521 Cesena, Italy. elena.babini2@unibo.it
authors.forename: Elena
authors.initials: E
authors.lastname: Babini
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Hu
authors.forename: Giacomo
authors.initials: G
authors.lastname: Parigi
authors.forename: Massimiliano
authors.initials: M
authors.lastname: Vignali
mesh_terms: D000595:Amino Acid Sequence; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002847:Chromatography, Agarose; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding
chemical_list: D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; C115911:EDF1 protein, human