We propose a series of biophysical studies on rhodopsin and the photoreceptive rod and cone outer segments (ROSs, COSs) of vertebrate retinas. The long range goal is a detailed understanding of the structure and function of the primary photoreceptor cells of the veterbrate visual system. 1) The structure of rhodopsin will be determined using lowdose electron microscopic (EM), surface shadowing and correlated x-ray diffraction techniques. Two-dimensional projections and 3-dimensional reconstructions will be derived from single-layered 2-dimensional crystals of frog rhodopsin initially; later studies will include bovine and toad rhodopsins. Chemical modifications of rhodopsin will be correlated with available primary sequence data to aid in interpreting the density maps. The production of 3-dimensional rhodopsin crystals will be pursued. 2) Scanning microinterferometry, scanning EM, freeze-fracture, freeze-substitution, histochemistry and microdensitometry of isolated ROSs from light-cycled frog and Xenopus will be employed to elucidate the structural bases of the birefringence banding and gradient in amphibian ROSs. Results will be correlated with known and suspected aspects of ROS disk renewal. 3) The structure, development, spacial relationships and comparative morphology of the ROS disk renewal. 3) The structure, development, spacial relationships and comparative morphology of the ROS disk perimeter and terminal loops will be studied by thin sectioning, freeze-fracture, freeze drying, surface shadowing and scanning EM techniques. The corresponding regions of COS disks will then be studied to establish similarities/differences. Our goal is to secure insights into the function(s) of these specialized regions of the fisk. 4) Determine the mass and apparent molecular weights of soluble proteins in frog ROSs, and determine the effects of bleaching on the binding of such proteins to isolated disk membranes. Correlated x-ray diffraction experiments on isolated ROSs and intact retina slices will be carried out to document the effects of bleaching on the diffraction patterns and electron density maps, changes in which will be used to more closely bracket the extent of bleach-induced protein binding to the disks under conditions closer to those in vivo.
