Progesterone-binding globulin interaction with its steroid ligands; study of the protein binding site topography using spin labeled steroids and electron spin resonance spectroscopy

Abstract

The binding site topography of progesterone-binding globulin (PBG) purified from pregnant guinea pig serum was examined using synthesized spin-labeled ligands and electron spin resonance (ESR) spectroscopy. A series of deoxycorticosterone-nitroxide (DOC-NO) derivatives were prepared, bearing the free radical on the side chain at increasing distance (d) from the steroid nucleus. The ability of the spin-labeled steroids to specifically bind to PBG was assessed by measurement of their relative binding affinity as compared to progesterone. ESR spectra of the bound steroid nitroxide radical were used to calculate the rotational correlation times tau c for the nitroxides as a function of their distance d to the protein-bound steroid nucleus. The data showed that the side chain nitroxide exhibited an unrestrained rotation in a water-like environment when d reached about 18 A. This would correspond to a PBG steroid binding site depth of about 28 A and suggests that the bound steroid in the PBG site is oriented with the side chain at C-17 directed toward the outside of the protein binding crevice.