Use of azidophenothiazines to study binding in proteins and in protein-protein interactions

The utility of photoaffinity reagents that contain an azidophenothiazine and a biotinyl moiety was recently demonstrated in a study of the hydrophobîc binding sites in the regulatory protein calmodulin (CaM) (DeLaLuz. et al.. Bioconjugate Chem. 1995, 6, 558). We now report that these azidophenothiazines also label the CaM-regulated protein phosphatase. calcineurin (Cn) and that the presence of CaM has a marked influence on Cn labeling. It has been suggested that the inhibition of Cn activity only involved phenothiazine binding to CaM. Our results demonstrate that azi do phenol hi azines bind directly to and photolabel Cn in its catalytic A subunit. In addition, complexation of Cn with CaM enhances this labeling. Photolabeling of the Cn/CaM complex using a bidentate reagent with two tethered azidophenothiazines produces a band at the combined apparent molecular weight of Cn A plus CaM. In a recent report, Cn was photolabeled by a photoactive cyclosporin A (CsA) derivative in the regulatory B subunit. We report here that azidopine, a compound that labels P-glycoprotein at nmol concentration, also photolabels Cn in the B subunit. CsA and ascomycin were determined to enhance the photolabeling of Cn with the azidophenothiazine but had no effect on the labeling with azidopine. Studies are currently in progress to determine the effect of photolabeling on the phosphatase activity of Cn. (supported by NIH grant GM 47427 to DSW).