Molecular localization of frog retinal receptor photopigment by electron microscopy and low-angle X-ray diffraction

Abstract

Low-angle X-ray diffraction patterns were obtained from ordered ultracentrifugal pellets of wet receptor disk membranes which had been either treated with antirhodopsin serum, normal rabbit serum, serum albumin, or untreated prior to sedimentation. A preliminary analysis of these patterns indicated: (a) differences between antirhodopsin serum treated and untreated preparations are due to conjugation of antirhodopsin molecules with their antigen in the disk membranes and not non-specific adsorption of other serum proteins to the disk membranes, (b) the planar ordering of the adsorbed antirhodopsin molecules over the surface of the disk membrane is nearly identical to that of the 40 to 50 A particles in the untreated disk membrane.A detailed Fourier analysis of these patterns in terms of a planar liquid-like arrangement of the 40 to 50 A particles in the untreated disk membranes and the antirhodopsin molecules adsorbed to the antirhodopsin serum treated disk membranes confirmed our preliminary analysis. The planar liquid-like arrangement of the 40 to 50 A particles is nearly identical to that of the adsorbed antirhodopsin molecules (3.0 and 3.1 nearest neighbors at a separation of 56 and 58 A respectively at 26 [deg] +/- 0.2 deg.C). Thus, the 40 to 50 A particles of the wet untreated disk membranes are the photopigment molecules.Electron micrographs of phosphotungstate negatively-stained disk membrane demonstrate particles ~40 A in diameter within the disk membrane. Optical transforms of these electron micrographs show that these particles appearing in the micrograph are arranged in a planar square array with a unit cell side of ~ 70 A. Correlation of these results with those obtained by low-angle X-ray diffraction in ultracentrifugal pellets of phosphotungstate stained and dried disk membranes as well as on wet pellets of untreated disk membranes before, during and after drying indicates the following: the ~ 40 A diameter particles seen in the electron micrographs are most likely the same 40 to 50 A particles giving rise to the observed low-angle X-ray diffraction from wet disk membranes. Hence the particles seen in the electron micrographs are most likely the nonpolar cores of the photopigment molecules.