An alternative assay to discover potential calmodulin inhibitors using a human fluorophore-labeled CaM protein

Fluorescence, circular dichroism, NMR, and docking studies of the interaction of the alkaloid malbrancheamide with calmodulin

“…In the first case, fluorescence titration curves for CaM with alkaloids 1−4 were constructed using a fluorescent-engineered hCaM (hCaM-M124C-mBBr) biosensor, according to the protocol previously described by González-Andrade et al 15 . For this, Ca 2+ -CaM was titrated with different amounts of compounds 1−4 and the fluorescence changes were monitored in the range between 400 and 550 nm, being the maximum response at 470 nm.…”

“…Uniformly 15 N and 13 C labelled hCaM were prepared in a salts minimal M9 medium containing 15 NH 4 Cl (0.5 g/L) and 13 C 6 -glucose (1.5 g/L), following the same procedure as for the unlabelled protein.…”

“…All experiments were run in the Varian Innova 500 instrument equipped with a 1 H, 13 C, 15 N TXI probe, by using sample tubes with a 5 mm outer diameter. Two-dimensional NMR spectra were recorded in the phase-sensitive mode using the States-TPPI method, and were carried out using the WATERGATE pulse sequence or excitation sculpting for water suppression to eliminate solvent signal in H 2 …”

“…Unlabelled and fluorescent-engineered human calmodulin (hCaM) were modified, over-expressed, and purified from Escherichia coli strain BL21-A1, as previously described by González-Andrade et al 15 . Uniformly 15 N and 13 C labelled hCaM were prepared in a salts minimal M9 medium containing 15 NH 4 Cl (0.5 g/L) and 13 C 6 -glucose (1.5 g/L), following the same procedure as for the unlabelled protein.…”

Fluorescent Biosensors for Protein Interactions and Drug Discovery

“…A mutant of CaM coupled to three different environment-sensitive fluorophores (MDCC, acrylodan, and IANBD ester) was detected the CaM interaction with phenothiazines and related tryclic antidepressants (Douglass, Salins et al 2002). Recently Gonález-Andrade and col, has been designed a alternative biosensing assay for CaM inhibitors by chemical modification of bromobimane at position 124 (Gonzalez-Andrade, et al 2009;Figueroa, et al 2010), that allowed to determine the IC 50 …”