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-biotinyl tosylate, inhibited the CaM-mediated activation of phosphodiesterase (PDE) with an I50 of 12.5 (+/- 2.8) microM. 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.
DeLaLuz
P J
PJ
Department of Chemistry, University of Kentucky, Lexington 40536, USA.
Golinski
M
M
Watt
D S
DS
Vanaman
T C
TC
D000345:Affinity Labels / Q000138:chemical synthesis* / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D004789:Enzyme Activation; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010640:Phenothiazines / Q000138:chemical synthesis* / Q000737:chemistry; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010777:Photochemistry
D000345:Affinity Labels; D002147:Calmodulin; D010640:Phenothiazines; D001710:Biotin; D010727:Phosphoric Diester Hydrolases