Mark Yeager, Benno P. Schoenborn, Donald M. Engelman
Mar 5, 1980
Journal of molecular biology
Abstract Neutron diffraction data have been collected from samples containing ten darkadapted Rana catesbiana bullfrog retinas in 100, 80, 60, 40 and 30% D 2 O † Ringer's solution using a step-scanning Soller slit diffractometer. Diffraction was also recorded from retinas equilibrated in D 2 O solutions with varying osmolarity. Rocking curve experiments demonstrated that the rods are disoriented in a cylindrically symmetrical fashion. Structure factor amplitudes were obtained using semi-automated curve-fitting procedures, and phases were obtained by interpreting the D 2 OH 2 O and osmotic Patterson maps. In D 2 O Ringer's solution the first four structure factors are −353 ± 25, 246 ± 19, 434 ± 13 and 383 ± 19. Neutron scattering density profiles were calculated to 75 A resolution using these structure factors. These neutron diffraction data are consistent with the view that the lipid bilayer is a major structural motif of the rod outer segment disc membrane. Neutron Fourier syntheses in different mixtures of D 2 O and H 2 O indicate that the intradisc and extradisc spaces are predominantly aqueous, consistent with the increase in the intradisc and extradisc volumes as the Ringer's solution is made more hypotonic. In isotonic Ringer's solution, the thicknesses of the intradisc and extradisc spaces are about 36 A and 160 A, respectively, and the center-tocenter separation between the 50 A thick lipid bilayers is 88 A. Assuming that the intradisc space is occupied by pure Ringer's solution, the maximum contrast match point for the hydrocarbon region of the disc membrane is 0.34 × 10 −14 cm/A 3 , corresponding to 13% D 2 O. If the 30 A thick hydrocarbon region is occupied exclusively by anhydrous protein and hydrocarbon, then the estimated maximum fraction of rhodopsin in the hydrocarbon region is only ~20%. Neutron scattering density profiles in D 2 O Ringer's solution are strikingly asymmetric with a scattering density on the extradisc side of the disc membrane lower than that of the intradisc space. Models that orient rhodopsin asymmetrically on the cytoplasmic face of the disc membrane extending into the extradisc aqueous space are suggested by the observed asymmetry. However, rhodopsin only accounts for roughly a third of the observed asymmetry, suggesting that a substantial amount of hydrogenated material resides in the extradisc region. This material may account for the stabilization of the regular, parallel arrangements of dises in the rod outer segment.