Synthesis and Use of a Biotinylated 3-Azidophenothiazine to Photolabel Both Amino- and Carboxyl-Terminal Sites in Calmodulin

The biotinylated probe, 3-azido-10-(4-(4-biotinyl-1-piperazinyl)butyl)phenothiazine, was used to examine the phenothiazine binding domains in calmodulin (CaM) by photolabeling. This phenothiazine, synthesized from 3-azido-10-(4-(1-piperazinyl)butyl)phenothiazine and d-biotiny 1 tosylate, inhibited the CaM-mediated activation of phosphodiesterase (PDE) with an I₅₀of 12.5 (±2.8) μM. Photolabeling of CaM produced covalent adducts in excellent yield (32%) in a light- and Ca²⁺-dependent manner. Studies performed over a range of drug concentrations suggested a 2:1 stoichiometry for the binding of the phenothiazine probe to CaM. Limited trypsin digestion and purification of the resulting fragments by either SDS-PAGE or HPLC provided two principal phenothiazine-containing peptides. Amino acid composition and sequence analyses performed on these two peptides established that both the N- and C-terminal domains in CaM, particularly the regions amino terminal to Ca²⁺-binding loops 1 and 3, were modified by the photoactivated phenothiazine derivative. These data, particularly for the C-terminal domain, are in excellent agreement with the X-ray structure analysis of a 1:1 CaM-trifluoperazine complex.