Physical analysis of light-scattering changes in bovine photoreceptor membrane suspensions.

We have used electron microscopy and model calculations to analyze the physical basis of light-scattering signals from suspensions of photoreceptor membranes. These signals have previously been used to probe interactions between photoactivated rhodopsin (R*) and the peripheral membrane enzyme, GTP-binding protein (G) (Kühn et al., 1981, Proc. Natl. Acad. Sci. USA., 78:6873-6877). Although there is no unique physical interpretation of these signals, we have shown in this study that they were qualitatively unchanged when the rod outer segment fragments (containing stacked disks) were fragmented by sonication or osmotic shock to produce spherical disk membrane vesicles. An exact treatment of the scattering process for spherical vesicles enabled us to evaluate the effects of changing membrane thickness, refractive index, or vesicle diameter. We present a particular redistribution of mass upon R*-G interaction that fits the experimental data.

Squid rhodopsin and GTP-binding protein crossreact with vertebrate photoreceptor enzymes.

The activation of photoreceptor GTP-binding protein by rhodopsin was studied in squid photoreceptors and in crossreactions between the squid and bovine proteins. Turbidity changes were observed in the far-red after photoexcitation of rhodopsin with brief flashes and were used to probe interactions between photoreceptor membrane suspensions and soluble protein extracts. Our findings are squid photoreceptors contain a GTP-binding protein detectable by light- and GTP-sensitive turbidity changes and by limited sequence homology of a 46-kilodalton polypeptide to the alpha-subunit of bovine GTP-binding protein; the squid membranes activate bovine GTP-binding protein qualitatively in the same way as bovine rhodopsin; the 46-kilodalton component is present in a membrane-bound fraction but is more abundant in a crude, soluble fraction of squid rhabdomes, and this soluble fraction can interact with either squid or bovine rhodopsin-containing membranes; light-activated GTPase activities in all of these preparations are consistent with the light-induced turbidity changes. These results show that rhodopsin activation of GTP-binding protein is highly conserved in vertebrate and cephalopod photoreceptors. Since squid rhodopsin is immobilized in precisely ordered microvilli, this suggests that activation of GTP-binding protein in cephalopod photoreceptors occurs in the absence of rhodopsin diffusion. The rhodopsin immobility may be compensated by higher mobility of the soluble GTP-binding protein.

Light-induced interaction between rhodopsin and the GTP-binding protein. Metarhodopsin II is the major photoproduct involved.

We have previously described [H, Kühn et al. (1981) Proc. Natl Acad. Sci. USA, 78, 6873-6877] a light-induced scattering change ('binding signal') associated with a stoichiometric binding between photoexcited rhodopsin and a peripheral membrane protein, the GTP-binding protein, in bovine rod outer segment suspensions. We have attempted here to identify the rhodopsin intermediate R* which is responsible for this interaction, by studying its dependence on pH, temperature and ionic strength. The results strongly suggest that the active state is metarhodopsin II (M II). 1. The initial phase of the binding signal is slightly slower than the formation of metarhodopsin II (2-37 degrees C, pH 5.5-9). 2. The kinetics of the decay of the active rhodopsin state are similar to those of the metarhodopsin II leads to metarhodopsin III transition (37 degrees C, pH 7.3). 3. All conditions which lead to light-induced binding of the GTP-binding protein to R* also lead to the formation of M II. At 2 degrees C, pH 8.3, in particular where no M II is formed in the absence of GTP-binding protein, binding signals and light-induced attachment of the GTP-binding protein to the membrane are still observed. Consistently, addition of GTP-binding protein to a suspension of extracted membranes bleached at 2 degrees C (pH 8.3) shifts the metarhodopsin I in equilibrium metarhodopsin II equilibrium towards metarhodopsin II. The shift is reversed by GTP, which dissociates the rhodopsin--GTP-binding protein complex. 4. At low ionic strength, where the GTP-binding protein is soluble in the dark (instead of being associated to the membrane as in the above experiments) M II still induces the binding whereas M I does not, indicating a much lower affinity of the GTP-binding protein for MI.

Orientational changes of the absorbing dipole or retinal upon the conversion of rhodopsin to bathorhodopsin, lumirhodopsin, and isorhodopsin.

The orientational change of the absorbing dipole of the retinal chromophore in vertebrate rhodopsin (rhodo) upon photo-excitation to bathorhodopsin (batho), lumirhodopsin (lumi) and isorhodopsin (iso), has been studied by polarized absorption and linear dichroism measurements on magnetically oriented frog rod suspensions that were blocked at liquid nitrogen temperature. Both the azimuthal component delta theta and the polar component delta theta of the total angular change were studied in separate experiments. Delta theta was estimated from polarized absorption measurements on rods oriented transversally with respect to the analyzing beam. The data show unequivocally that upon the rhodo leads to batho transition, the dipole shifts out of the membrane plane by only few degrees; delta theta congruent to -3 degree. This azimuthal shift was nearly exactly reversed upon the batho leads to lumi decay. A very small shift (delta theta less than or equal to 1 degree) toward the membrane plane was observed upon a rhodo leads to iso conversion. The polar component delta theta of the angular shift was estimated by studying the photoreversion of linear dichroism induced by photo-excitation with polarized light in rods oriented parallel to the analyzing beam. Upon the rhodo leads to batho transition, ther was a shift delta theta = 11 +/- 3 degrees. The overall angular shift upon this first photo-exciting step, which corresponded to the isomerisation of retinal, was only delta omega = 11 +/- 3 degrees. This is smaller than what may be expected for a cis-trans isomerization of a retinal molecule with one end fixed, and different from what has been previously estimated by another group. These discrepancies are discussed.

Interactions between photoexcited rhodopsin and GTP-binding protein: kinetic and stoichiometric analyses from light-scattering changes.

In rod outer segments, photoexcited rhodopsin (R*) activates a cyclic GMP phosphodiesterase through a sequence of reactions involving a GTP-binding protein. By measuring light-scattering changes above 700 nm, we have studied the kinetics and stoichiometry of the association of R* with this protein and of the dissociation of the complex upon GDP/GTP exchange. Two light-scattering signals were obtained upon photoexcitation of rhodopsin in bovine rod outer segment membranes as well as in a reconstituted system consisting of purified GTP-binding protein and washed disc membranes; both signals depended specifically on the presence of GTP-binding protein. A "binding signal" that was observed in the absence of gTP as an increase in turbidity became saturated when a number of rhodopsin molecules equal to the number of GTP-binding protein molecules present (congruent to 10% in rod outer segments) has been bleached, suggesting that the protein binds to R* in a 1:1 complex. A "dissociation signal" of opposite sign, observed in presence of GTP at greater than or equal to 1 microM, is half maximal at 0.04% bleaching and saturated at 0.5% bleaching; it is interpreted as reflecting the dissociation of GTP-binding protein-R* complexes after GDP/GTP exchange on the GTP-binding protein, one R* being able to interact sequentially with about 100 GTP-binding protein molecules. The early time course of the binding signal is faster than that of the dissociation signal, and both signals take place in the 100-msec range at 20 degrees C.

Orientation of emitting dipoles of chlorophyll A in thylakoids: considerations on the orientation factor in vivo.

Orientation angles of five emitting dipoles of chlorophyll a in thylakoids were estimated from low temperature fluorescence polarization ratio spectra of magnetically oriented chloroplasts. A simple expression is given also for the evaluation of data from linear dichroism measurements. It is shown that the Qy dipoles of chlorophylls lie more in the plane of the membranes and span a larger angular interval than was previously thought. Values for the orientation factor are calculated using various models corresponding to different degrees of local order of the Qy dipoles of chlorophylls in the thylakoid. We show that the characteristic orientation pattern of the Qy dipoles of chlorophylls in the membrane, i.e., increasing dichroism toward longer wavelengths, may favour energy transfer between the antenna chlorophylls as well as funnel the excitation energy into the reaction centers.

Cyanine dye measurement of a light-induced transient membrane potential associated with the metarhodopsin II intermediate in rod-outer-segment membranes.

The fluorescent dye 3,3'-dipropylthiodicarbocyanin iodide [diS-C3-(5)] is used as a probe of fast membrane potential variations in the study of rod-outer-segment membranes. The formation of metarhodopsin II is found to be associated with the generation of a transmembrane potential, which is dissipitated by ionic movements across the membrane; its risetime is very similar to that of the protonation of the protein, which is also associated with the formation of metarhodopsin II.

Orientation of rhodopsin alpha-helices in in retinal rod outer segment membranes studied by infrared linear dichroism.

Frog retinal rod outer segments, oriented by a magentic field, were shown to contain rhodopsin alpha-helical segments preferentially aligned perpendicular to the plane of the disc membrane, by the technique of infrared linear dichroism. Infrared absorption parallel and perpendicular to the rod axes by peptide C parallel to O groups, whose absorption band contains alpha-helical and random coil components at slightly different frequencies, showed positive dichroism centered on the alpha-helix frequence. We conclude that the alpha-helical portion of the protein has an average orientation in the transmembrane direction. Furthermore, infrared spectra of rods in 2H2O Ringer's solution exhibit two distinct peptide amino group absorption bands: the unexchanged N-2H band, which is nondichroic. This implies that the oriented part of the protein is in the lipid bilayer, supporting a model for rhodopsin with a hydrophobic core containing partially oriented alpha-helices and hydrophilic ends consisting of unoriented polypeptide.