STRUCTURE OF VISUAL PIGMENTS .2. BINDING OF RETINAL AND CONFORMATIONAL CHANGES ON LIGHT EXPOSURE IN BOVINE VISUAL PIGMENT500

Previous observations (Bownds, D., and Wald, G. (1965), Nature 205,254; Wald, G., and Brown, P. K. (1952), J. Gen. Physiol. 35, 797) showing that while native visual pigment is not susceptible to reduction with sodium borohydride, light-exposed visual pigment can be reduced with borohydride to produce a retinyl lysine derivative of visual pigment, and that only after exposure to light are free sulfhydryl groups detectable in visual pigment, have been quantitatively extended to show that purified bovine visual pigment500 contains one retinal chromophore per molecule and that only one sulfhydryl group per molecule becomes titrable on exposure to light. The four other half-cystine residues in visual pigment are present as two disulfide bridges. Two conformational forms of visual pigment have been separated by gel filtration chromatography: a compact conformation shown by native visual pigment and an expanded conformation shown by the apoprotein. The light-exposed, borohydride-reduced visual pigment, in which retinal is covalently bound to the protein, also shows the expanded conformation. The Stokes radius of the native pigment is 23 Å, that of the apoprotein and the light-exposed, borohydride-reduced pigment is 25.5 Å, and their hydrodynamic volumes are in the ratio of 1:1.36. On the basis of these experiments it is proposed that the 11-cis isomer of retinal is bound to a unique lysine residue in native visual pigment through a substituted aldimine bond. The substituting moiety is a cysteine sulfur. The substituted aldimine bond functions as a cross-linking agent between a lysine and a cysteine residue, maintaining the native molecule in a compact conformation. Two photochemical reactions take place in the excited-state molecule: the C-S bond is broken and retinyl isomerizes to the all-trans form (Wald, G., and Hubbard, R. (1960), Enzymes 3, 369). This leads to a reversion of the substituted aldimine to a simple aldimine bond (making the linkage susceptible to borohydride reduction) and exposing a new titrable sulfhydryl group. This reversion of the aldimine bond breaks the cross-linkage between the lysine and cysteine residues thus transforming the molecule into its expanded conformation. It is further proposed that functioning as cross-linking agent is a general property of substituted aldimine bonds in proteins and that other cases of compact-expanded conformational transformations can be expected in this class of proteins. © 1968, American Chemical Society. All rights reserved.