ORIENTATION OF AROMATIC RESIDUES IN RHODOPSIN. ROTATION OF ONE TRYPTOPHAN UPON THE META I→META II TRANSITION AFTER ILLUMINATION

Abstract— Linear dichroism measurements have been performed in the visible and the UV on suspensions of intact retinal rod outer segments, isolated from frog and from cattle retinas and oriented by a magnetic field. In the UV a sharp double peaked signal is observed around 290 nm. It is characteristic of a tryptophan residue having its ¹L_b transition oriented preferentially perpendicular to the membrane plane. Only one tryptophan residue per rhodopsin molecule seems to be involved, and this is insufficient to account for the diamagnetic anisotropy of the rods which causes their orientation. Upon bleaching one observes a rotation of this tryptophan coupled to the Meta I →Meta II transition, and the rotation is reversed in the next step Meta II → Meta III. The correlation with other spectral changes suggests that this tryptophan is in close relation with the retinal chromophore.     

Porphyrins as photosensitizers to enhance night vision

Relative bleaching rates of bovine rhodopsin (rod outer segments) in the presence and absence of seven porphyrins and methylene blue were measured under exposure to lambdamax = 675 nm light, using UV-vis spectroscopy. Rate enhancements on the order of up to three times compared to the bleaching of rhodopsin alone where observed. Fluorescence measurements and other data suggests that the porphyrins act as photosensitizers and excite the visual pigment via electron or triplet state energy transfer. These mechanisms suggest that rhodopsin possesses a pocket, proximal to the Schiff base so that porphyrins act as photosensitizers.     

EVIDENCE FOR A MAGNESIUM DEPENDENT ATPase IN BOVINE ROD OUTER SEGMENT DISK MEMBRANES*†

Abstract—In the presence of Mg²⁺ and adenosine triphosphate (ATP), a rapid. light-induced, light-scattering transient is observed from bovine rod outer segments (ROS). This light-scattering transient we have labelled 'A'. Ca²⁺ cannot replace Mg²⁺. nor can guanosine triphosphate (GTP) replace ATP. 'A' is observed at ATP concentrations as low as a few μM.
The half-time of 'A', 60 ms at 20° and 20 ms at 37°, is consistent with a process possibly involved in visual transduction.
'A' has the action spectrum of rhodopsin bleaching and its amplitude is strictly proportional to the fraction of rhodopsin bleached per flash. 'A' can be regenerated by 11- cis retinal.
Inhibition studics with ATP analogues, which cannot be hydrolysed and fail to evoke an 'A' response, reveal that an ATP hydrolysis process has to precede illumination in order for 'A' to occur.
On the basis of the above findings. it is proposed that there is a Mg²⁺ dependent ATPase in ROS that allows the disk membrane to assume a new membrane state which, upon illumination, is altered. giving rise to the structural phenomenon monitored as light-scattering transient 'A'.     

ORIENTATION OF RETINYLIDENE CHROMOPHORE OF HYPSORHODOPSIN IN FROG RETINA

Abstract— Hypsorhodopsin and bathorhodopsin were formed in the frog retina by irradiating rhodopsin at liquid He temperature (9 K) with orange light (> 520 nm) and blue light (437 nm), respectively. Hypsorhodopsin was converted to bathorhodopsin in the retina by warming above 32 K in the dark. Similar phenomena were observed in the rod outer segment suspension. A difference spectrum between hypsorhodopsin and bathorhodopsin in the retina produced by warming was almost identical with that in the rod outer segment suspension. This suggests that the transition dipole moment of hypsorhodopsin is parallel to the disk membrane plane which is also parallel to that of bathorhodopsin.     

LIGHT-INDUCED PERTURBATION OF AROMATIC RESIDUES IN BOVINE RHODOPSIN AND BACTERIORHODOPSIN*

Light-induced changes in the UV absorption spectrum of bovine rod outer segment membranes were measured by conventional difference spectroscopy and by flash photolysis methods. Different thermal intermediates of rhodopsin (lumirhodopsin, metarhodopsin I, metarhodopsin II, and meta-rhodopsin III) have absorption spectra in the ultraviolet which differ from the rhodopsin spectrum and from each other. The spectra associated with metarhodopsin I, metarhodopsin II, and metarhodopsin III are characteristic of perturbation of a small number of tyr. and/or trp residues, most likely one trp residue. These aromatic residues are in the neighborhood of the retinal Schiff base and undergo coordinated changes of interaction with retinal during the bleaching sequence. At the metarhodopsin II stage, the magnitude of the UV spectral changes is consistent with the exposure of a previously shielded trp residue to an aqueous environment. The present results are consistent with previous spectral studies which limit the extent of light-induced conformational changes to regions of the protein in the neighborhood of the retinal Schiff base. An analogous study was made on light-adapted purple membranes of Halobacterium halobium. The UV absorption spectrum associated with the deprotonated Schiff base intermediate of the trans-bacteriorhodopsin cycle is indicative, in part, of aromatic residue perturbation. However, significant changes in the secondary and tertiary structures of the bacterio-rhodopsin protein characteristic of a delocalized conformational change are unlikely at this intermediate stage.     

THE CIRCULAR DICHROISM OF SODIUM CHOLATE SOLUBILIZED RHODOPSIN

Abstract— The near UV and visible circular dichroism (CD) spectra of rhodopsin solubilized and purified in sodium cholate have been determined. The CD properties of rhodopsin in 2 and 20mg/ml sodium cholate are substantially different in terms of the α band to β band ratio, and sensitivity of the near UV CD spectra to bleaching. Rhodopsin in 2mg/m l sodium cholate will regenerate (11- cis -retinal + opsin → rhodopsin) and has a CD spectrum similar to rhodopsin in rod outer segment membranes and digitonin which will also regenerate. On the other hand rhodopsin in 20mg/m l sodium cholate will not regenerate and has CD properties similar to other nonregenerable detergents (cetyltri-methylammonium salts and emulphogene). These results indicate that CD reflects the conformational integrity of functional (regenerable) rhodopsin and that sodium cholate can reversibly alter the conformation of rhodopsin. Finally the results further support the validity of using sodium cholate solubilized rhodopsin as a model system for studies on the structure and function of rhodopsin.     

Rhodopsin phosphorylation suggests biochemical heterogeneities of retinal rod disks.

Frogs (Rana pipiens) were injected subcutaneously with (3H)-leucine and allowed to incorporate the radioactive amino acid into newly assembled disks in the retinal rod outer segment. The labeled disks served as a temporal marker for following the turnover of rod outer segments. Animals were killed at different times after injection and outer segments were isolated and phosphorylated with ATP in the light. The visual pigment (as isorhodopsin) was regenerated with 9-cis retinal, extracted, and chromatographed on epichlorohydrin triethanolamine cellulose so that phosphorylated pigment could be separated from unphosphorylated pigment. The ratio of (3H)-radioactivity of phosphorylated pigment to that of unphosphorylated pigment was then plotted against the time after injection. The ratio was high when (3H)-labeled disks were largely associated with the basal region of the rod and decreased as the labeled disks moved toward the rod apical region. The results were interpreted as suggesting that newer disks are phosphorylated preferentially to older disks. Papain digestion of (3H)-labeled disks indicated that rhodopsin in newer disks is more susceptible to proteolysis than that in older disks.     

INCORPORATION OF 11,12-DIHYDRORETINAL INTO THE RETINAE OF VITAMIN A DEPRIVED RATS

Abstract— The incorporation of 11,12-[15–³H]-dihydroretinal, a retinal in which the crucial 11-ene is saturated, into the retinae of vitamin A deficient rats as a result of intraperitoneal injection of the corresponding alcohol was shown by the presence of the tritium label in the rod outer segments and by identification of the extracted retinals using high pressure liquid chromatography. The amplitude of the electroretinogram (ERG) b-wave, diminished as the result of vitamin A deprivation, was not affected by administration of the analogue, although similar treatment of deprived litter mates with trans retinal restored the ERG b-wave amplitude to a normal level.
The evidence that the analogue is bound to opsin forming 11,12-dihydrorhodopsin is as follows: (1) when incubated with 11- cis retinal, extracts from vitamin A deficient rats regenerate 1.4 nmol rhodopsin while extracts from rats deficient in vitamin A and supplemented with 11,12-dihydroretinal regenerate 0.6 nmol rhodopsin indicating binding of the dihydroretinal blocks rhodopsin regeneration. (2) 11,12-dihydroretinal is shown to remain unchanged in hexane-washed retinae after extraction with methylene chloride and (3) injection of retinal into animals previously injected with 11,12-dihydroretinal also fails to restore visual sensitivity as measured by the ERG b-wave. Our results indicate that the dihydro-chromophore occupies the same binding site as the natural 11- cis retinal and that occupation of the chromophore binding site of opsin is not sufficient to restore the visual sensitivity in a vitamin-A-deprived animal.     

PHOTODICHROISM OF RHODOPSIN SOLUTIONS AT -196°C

Abstract— The photodichroism of a system of randomly oriented, bleachable pigment molecules, rigidly fixed in an inert transparent matrix was investigated theoretically. A formula was derived for the photochemical equilibrium reaction between three pigments, N ₁⇆ N ₂⇆ N ₃ in the case that the absorption ellipsoids of the pigments are rotationally symmetric and their symmetry axes coincide. The formula was applied to the dichroism induced by plane polarized light of different wavelengths in an aqueous rhodopsin-glycerol mixture at –196°C. It was found that the absorption ellipsoids of this visual pigment, its analogue, isorhodopsin, and its first bleaching product, prelumirhodopsin, are elongated with an apparent axial ratio of about 5. It was concluded that the light absorbing properties of these pigments cannot be described by a single transition moment vector (linear absorber). The absolute value of the quantum efficiency of the conversion of rhodopsin to prelumirhodopsin was shown to be approximately equal to the quantum efficiency of bleaching rhodopsin at room temperature. Some evidence was obtained that indicates that the relative quantum efficiencies of the rhodopsin system at – 196°C may be wavelength dependent.     

LIGHT-INDUCED CHANGE IN RHODOPSIN EMISSION: PHOSPHORESCENCE and FLUORESCENCE

Abstract— Phosphorescence measurements of rhodopsin in bovine rod disk membranes were made to study changes in protein conformation on bleaching by probing the environment of tryptophan and tyrosine residues of the protein. Bleaching decreased the tyrosine phosphorescence by about 25% and significantly affected the amplitude of triplet decay when rhodopsin was excited at 280 nm, where both tyrosine and tryptophan absorb. Computer analysis using one or two exponential model functions showed the presence of two components in the decay curve at 410 nm—one with a lifetime of 2.2 s, the other with a lifetime of 4.8 s>—which are typical of tyrosine and tryptophan respectively. When the rod outer segment sample was bleached, there was a significant decrease in the amplitude of the tyrosine component. However, the lifetime values of the two components did not change. Analyses of the fluorescence spectra of dark and bleached membranes at different excitation wavelengths and the phosphorescence change on bleaching suggest energy transfer between tyrosine and tryptophan singlet states, which may result from a conformational change of the opsin moiety on bleaching.     

Longitudinal diffusion of a polar tracer in the outer segments of rod photoreceptors from different species

Vertebrate rod photoreceptors are the ultimate light sensors, as they can detect a single photon. In darkness, rods maintain a high concentration of the intracellular messenger cyclic guanosine monophosphate (cGMP), which binds to and keeps open cationic channels on the plasma membrane of the outer segment. Absorption of a photon by the visual pigment of the rod, rhodopsin, initiates a biochemical amplification cascade that leads to a reduction in the concentration of cGMP and closure of the channels, thereby converting the incoming light to an electrical signal. Because the absorption of a photon and the ensuing reactions are localized events, the magnitude of the response of the rod to a single photon depends on the spread of the decrease in the cGMP concentration along the length of the outer segment. The longitudinal diffusion of cGMP depends on the structural parameters of the rod outer segment, specifically the area and the volume available for diffusion. To characterize the effect of rod outer segment cytoarchitecture on diffusion, we have used fluorescence recovery after photobleaching (FRAP) and examined the mobility of a fluorescent polar tracer, calcein, in the rod outer segments from three species with different outer segment structures: frog (Rana pipiens), mouse (Mus musculus domesticus) and gecko (Gekko gekko). We found that the diffusion coefficient is similar for all three species, in the order of 8-17 microm(2) s(-1), in broad agreement with the predictions by Holcman and Korenbrot (Biophys. J. 2004:86;2566-2582) based on the known cytoarchitecture of rod outer segments. Consequently, the results also support their prediction that the longitudinal spread of light excitation in rods is similar across species.     

DISTRIBUTION OF ENZYMES INVOLVED IN NUCLEOTIDE METABOLISM IN THE DISK AND OTHER ROD MEMBRANES

Abstract— Intact disks and inverted disks were prepared from bovine retinal rods and the distribution in the disk membrane of such enzymes as guanyl cyclase, cyclic nucleotide phosphodiesterase, GTP binding protein (GTPase), 5'-nucleotidase and rhodopsin kinase was investigated. Guanyl cyclase was not detected in the disk; the enzyme activity was high in a membranous fraction containing the cilium or axoneme and the rod outer segment plasma membrane. Cyclic nucleotide phosphodiesterase, GTP binding protein (GTPase) and rhodopsin kinase were associated on the external surface of disk in the presence of 2 m M Mg²⁺. The enzymes dissociated from the membrane when Mg²⁺ was depleted. Thus, magnesium ion seems to regulate the state of these enzymes in the outer segment. 5'-Nucleotidase activity was low in intact disks but was significantly enhanced after inversion of the disk. The catalytic site of the enzyme, therefore, must be located on the internal (intradiscal) surface. Since the disks are known to be formed by invagination of the plasma membrane, 5'-nucleotidase, by inference, would have its catalytic site exposed on the external surface of the plasma membrane. Preliminary experiments showed that the capability of light-activated rhodopsin to activate cyclic nucleotide phosphodiesterase was inhibited by phosphorylation of the pigment. This supports the idea that rhodopsin kinase, cyclic nucleotide phosphodiesterase and GTPase exist as a functional complex on rod membranes.     

INTERPRETATIONS OF THE SOLUTION AND ORIENTED FILM SPECTRA OF BROWN MEMBRANE OF HALOBACTERIUM HALOBIUM

Abstract— The absorption and circular dichroic spectra of the brown holo-membrane (retinal present) and apo-membrane (retinal absent) of Halobacterium halobium in solution and oriented as a film have been studied over the accessible wavelength region, 800–183nm. Since the structure of the well-studied purple membrane can be considered to be a modification of the structure of the brown membrane and much is known about the structure of the purple membrane, interpretations of the brown membrane spectra are based on our previous interpretations of similar studies of the purple membrane. The brown membrane contains two membrane proteins, bacteriorhodopsin and cytochrome b-561 in a 3:1 molar ratio in contrast to the purple membrane which contains only bacteriorhodopsin. Main findings are (a) degenerate oscillator coupling (exciton) among the retinyl chromophores of the bacteriorhodopsins, (b) a relatively strong in-plane interaction between the retinal and the bacteriorhodopsin apoprotein environment, possibly due to a dissymmetric static charge distribution, (c) the planes of the aromatic rings of some of the tryptophans must be nearly parallel to the plane of the membrane, (d) the helical axes of the bacterio-opsin polypeptide segments are significantly tilted in respect to the normal to the membrane plane in contrast to the helical axes of the bacteriorhodopsin polypeptide segments which are nearly parallel to the normal, (e) no detectable interaction between the two membrane proteins, (f) the plane of the heme of the cytochrome cannot be parallel to the membrane plane and is most likely perpendicular to it. (g) the dipole moments of the two mutually perpendicular Soret porphyrin transitions of the heme are most likely oriented at an angle to the membrane plane, (h) there seems to be a significant reduction in the symmetry of the heme group in the environment of the apoprotein, (i) the possibility of a unique geometrical arrangement and resonance interaction between the Soret porphyrin and nearby cytochrome aromatic amino acid π–π* transitions, (j) the secondary structure of the cytochrome is significantly α-helical, and (k) the helical axes of the cytochrome polypeptide segments are randomly oriented in respect to the normal to the membrane plane. A consequence of these findings is that the fine structures of the bacteriorhodopsins of the brown and purple membranes are very similar in spite of differences in the composition and the structure of the two membranes. In addition, the orientation of the helical segments of the bacteriorhodopsin polypeptides relative to the membrane plane in the brown and purple membranes can be regulated by the retinal–apoprotein interactions. Significance of this possible regulation in respect to the proton-pumping function of these membranes is discussed.     

All‐Trans Retinal Mediates Light‐Induced Oxidation in Single Living Rod Photoreceptors†

All‐trans retinal is a potent photosensitizer that is released in photoreceptor outer segments by the photoactivated visual pigment following the detection of light. Photoreceptor outer segments also contain high concentrations of polyunsaturated fatty acids, and are thus particularly susceptible to oxidative damage such as that initiated by light via a photosensitizer. Upon its release, all‐trans retinal is reduced within the outer segment to all‐trans retinol, through a reaction requiring metabolic input in the form of NADPH. The phototoxic potential of physiologically generated all‐trans retinal was examined in single living rod photoreceptors obtained from frog (Rana pipiens) retinas. Light‐induced oxidation was measured with fluorescence imaging using an oxidation‐sensitive indicator dye from the shift in fluorescence between the intact and oxidized forms. Light‐induced oxidation was highest in metabolically compromised rod outer segments following photoactivation of the visual pigment rhodopsin, and after a time interval, sufficiently long to ensure the release of all‐trans retinal. Furthermore, light‐induced oxidation increased with the concentration of exogenously added all‐trans retinal. The results show that the all‐trans retinal generated during the detection of light can mediate light‐induced oxidation. Its removal through reduction to all‐trans retinol protects photoreceptor outer segments against light‐induced oxidative damage.     

THE STRUCTURAL CHANGES IN BOVINE ROD OUTER SEGMENTS IN THE PRESENCE OF ATP

Abstract— We have, previously, described a light-induced near infrared (700–850 nm) light scattering transient obtained in the presence of ATP from bovine rod outer segments suspensions in which the plasma but not the disk membranes were perforated (Uhl et al ., 1979a). This transient was termed the 'A' signal. To elucidate its possible origin, we have analyzed their angular and wavelength dependencies. These data have been compared with osmotically controlled (non-light) induced light scattering changes from identical control rod outer segments suspensions. It has been found that A_D (the dark light scattering signal obtained in the presence of ATP) and A_LS (the slow component of the actinic flash induced light scattering signal, A_L) can be assigned to the swelling of the disk membranes while A_Lf (the fast component of this latter signal) can be attributed to the change in refractive index of the ROS caused by the hypsochromic spectral shift of photolyzed rhodopsin. The collective disk swelling associated with A, and A_LS is consistent with the pumping of ions into the disk lumen by the action of a disk membrane bound ATPase.     

THE ULTRAVIOLET FLUORESCENCE OF BACTERIORHODOPSIN AND THE LOCATION OF TRYPTOPHANYL RESIDUES

Abstract— The ultraviolet fluorescence spectrum of bacteriorhodopsin is characterized by emission from an ensemble of internal, surface and exposed Trp residues. The temperature dependence of fluorescence yields exhibits a discontinuity at about 30°C coincident with previously observed transitions in membrane lipid microviscosity, photocycle lifetime and photoconductivity. Quenching at high pH coincides with ionization of Tyr and an emission red shift to a spectrum typical of that of tyrosinate. Guanidine hydrochloride produces only partial protein denaturation, increasing the number of exposed Trp by 50%. While exposed Trp in native bacteriorhodopsin are in the minority, they represent a higher proportion of total Trp than is found in rhodopsin of animal rod outer sections.     

FLASH PHOTOLYSIS OF ROD PARTICLE SUSPENSIONS

Abstract. Flash photolytic experiments with suspensions of rod particles isolated from cattle eyes reveal that at low temperatures a transient is formed following irradiation which preceeds lumirhodopsin in the bleaching process. Kinetic studies, in the range -75°C to -50°C, suggest that the transient is composed of three intermediate species, having similar absorption spectra but changing to lumirhodopsin at different rates. The energies of activation of the decay reactions appear to be identical and equal 12.5°3 kcal/mole. Spectrophotometric experiments have demonstrated that the low temperature transient is stable at liquid nitrogen temperatures and suggest that it can be reversibly converted to a mixture of rhodopsin and isorhodopsin at -195°C.
A detailed comparison of light initiated reactions of rhodopsin in rod particle suspensions and in solutions reveals striking similarity in the two systems.     

Biochemical steps in visual transduction: roles for nucleotides and calcium ions

Abstract— Recent studies from this laboratory permit the suggestion of a scheme for describing molecular mechanisms which may regulate excitation and adaptation in amphibian rod photoreceptor cells. The experiments have studied several chemical changes which occur upon illumination of isolated rod outer segments: (1) activation of cyclic GMP phosphodiesterase which is sensitive to calcium concentration, (2) a resulting rapid drop in cyclic GMP levels which has stoichiometry and time course appropriate for the internal transmitter presumed to mediate between photon absorption in the disc membrane system and the permeability decrease in the plasma membrane, (3) a dephosphorylation of two small proteins whose phosphorylation is controlled by cyclic GMP levels, and (4) a slower hydrolysis of GTP which may drive efflux of calcium from the outer segment. It is suggested that the rapid decrease in sodium conductance which follows illumination is caused by the dephosphorylation of the two small proteins, with their dephosphorylation being controlled by the cyclic GMP decrease. In a slower reaction light activates a GTP-dependent extrusion of calcium from the cytoplasmic space. This lowering of internal calcium causes desensitization of the light-sensitive phosphodiesterase enzyme responsible for the cyclic GMP decrease, so that its intensity-response function now resembles that of the light-adapted rod photoreceptor. Thus, changes in plasma membrane conductance are regulated by cyclic GMP, and the sensitivity of the system is controlled by slower calcium movements which set the light-sensitivity of the phosphodiesterase enzyme. Finally, the light-initiated phosphorylation of rhodopsin also appears to play a role, with phosphorylated rhodopsin causing desensitization of the phosphodiesterase enzyme.     

Light-regulated permeability of rhodopsin-phospholipid membrane vesicles.

Abstract—Light absorption by rhodopsin in receptor cell membranes initiates the excitation of the receptor cell. Rhodopsin-phospholipid membrane vesicles were studied to localize initial transduction events. Rhodopsin-phospholipid recombinant membranes are thermally stable and light sensitive and may be chemically regenerated after bleaching in the same manner as receptor cell membranes. Rhodopsin-containing vesicles prepared from unsaturated phosphatidylcholine (PCho) or PCho and phosphatidylethanolaminc display kinetics for the metarhodopsin I to II transition which are comparable to those of receptor cell membranes. NMR spectroscopy was used to examine the permeability of the membrane vesicles to added shift (Eu³⁺) or relaxation reagents (Mn²⁺, Co²⁺). Unexposed rhodopsin-phospholipid vesicles are sealed to ion movement and become permeable after light exposure. Selected ions (Ca²⁺, Mn²⁺, Co²⁺) may be photoreleased from the interior of loaded membrane vesicles. The quantity released is proportional to the initial ionic concentration. The number of ions released/rhodopsin bleached is dependent on the light intensity, and high yields (40–160) of Ca²⁺/rhodopsin bleached are observed at low levels of light bleaching. The present results indicate that rhodopsin spans the phospholipid bilayer membrane, and are consistent with an increase in the permeability of the membrane initiated by light excitation of rhodopsin.     

THE REGENERATION OF VISUAL PIGMENTS AND THE CHANGE OF ROD HYPERSENSITIVITY AFTER IRRADIATION BY BLEACHING LIGHT IN FROG RETINA

Abstract— The regeneration processes of visual pigments and the dark adaptation processes of rod photoreceptor after irradiation by bleaching light were studied by spectrophotometric, electroretinographic(ERG) methods and the measurement of early receptor potentials (ERPs) in bullfrog retina. After irradiation by bleaching light, rhodopsin in the isolated retina regenerated to an extent depending on the wavelength and intensity of the bleaching light as well as pH. Intense blue light and a weak alkaline environment (pH 7.5–9.5) favoured the regeneration. The regeneration of pigment in the green rods could not be detected in these experiments on the isolated retina. The regeneration of cone pigment was studied by measuring ERPs from both isolated retinas and retinas with pigment epithelium-choroid complex separated from scleras, which are called PEC-retinas. In the PEC-retinas, cone pigment regenerated more rapidly and with better efficiency than in the isolated retinas.
Rod photoreceptors desensitized permanently by bleaching light did not demonstrate hypersensitivity at 0.1 m M [Ca²⁺]_out, which induced hypersensitivity in non-desensitized photoreceptor, but showed the hypersensitivity when the [Ca²⁺]_out, was lowered further by the addition of EGTA.