Isomerization Of Rhodopsin Research Articles

The transition state in the isomerization of rhodopsin

We present an ab-initio molecular dynamics study of a model of the chromophore of rhodopsin. The 11- cis to all- trans ground-state isomerization of a retinylidene–ethylimine·HCl has been induced by applying an external classical force field to the C10–C11–C12–C13 dihedral angle, in addition to the ab-initio forces. By using this hybrid approach, we find that the transition state is characterized by a well-defined bond pattern along the chromophore and by a positive charge displacement. The relaxation of the chromophore is associated with propagation of a conjugation defect that transports the excess positive charge along the backbone.

The AII Amacrine Network: Coupling can Increase Correlated Activity

Retinal ganglion cells in the cat respond to single rhodopsin isomerizations with one to three spikes. This quantal signal is transmitted in the retina by the rod bipolar pathway: rod→rod bipolar→AII→cone bipolar→ganglion cell. The two-dimensional circuit underlying this pathway includes extensive convergence from rods to an AII amacrine cell, divergence from a rod to several AII and ganglion cells, and coupling between the AII amacrine cells. In this study we explored the function of coupling by reconstructing several AII amacrine cells and the gap junctions between them from electron micrographs; and simulating the AII network with and without coupling. The simulation showed that coupling in the AII network can: (1) improve the signal/noise ratio in the AII network; (2) improve the signal/noise ratio for a single rhodopsin isomerization striking in the periphery of the ganglion cell receptive field center, and therefore in most ganglion cells responding to a single isomerization; (3) expand the AII and ganglion cells' receptive field center; and (4) expand the “correlation field”. All of these effects have one major outcome: an increase in correlation between ganglion cell activity. Well correlated activity between the ganglion cells could improve the brain's ability to discriminate few absorbed external photons from the high background of spontaneous thermal isomerizations. Based on the possible benefits of coupling in the AII network, we suggest that coupling occurs at low scotopic luminances. Copyright © 1996 Elsevier Science Ltd.

Vibrational coherence effects on electronic curve crossing

Quantum dynamical simulations of vibrational phase coherence effects on ultrafast electronic curve crossing in condensed phases are presented for both symmetric and barrierless double well potentials. Reaction coordinate probability distributions are constructed from the reduced density matrix of the system, which provide considerable insight into the dynamics of curve crossing. The application of these ideas to retinal isomerization in rhodopsin is presented. Results suggest that coherence transfer processes are important for interpreting recent femtosecond transient absorption results on this system.

FTIR spectroscopy reveals microscopic structural changes of the protein around the rhodopsin chromophore upon photoisomerization.

Fourier transform infrared spectroscopy was used to investigate the local structure around the chromophore of rhodopsin and its change upon photoisomerization. A hydrated film of bovine rod outer segments was cooled at 80 K, and difference infrared spectra were obtained between bathorhodopsin and rhodopsin or between bathorhodopsin and isorhodopsin under suitable irradiation conditions. The spectra in a higher-frequency region (4000-1800 cm-1) revealed protein structural change by probing the O-H, N-H, and S-H stretching vibrational modes. The structural change of bound water molecules occurred upon formation of bathorhodopsin, where three water O-H increased the strength of their H-bonding. The water structure is identical in rhodopsin and isorhodopsin. These results suggest that the protein in the close proximity of the Schiff base of the chromophore is perturbed upon photoisomerization and causes rearrangement of the water molecules in bathorhodopsin. Upon the isomerization, the 3463 cm-1 band of the 11-cis form (rhodopsin) shifts to 3487 cm-1 for the all-trans form (bathorhodopsin) or to 3481 cm-1 for the 9-cis form (isorhodopsin). An N-H bond, possibly of an indole of tryptophan residue, is responsible for these bands. It is present in a hydrophobic environment around the beta-ionone ring and/or polyene chain of the retinal, and changes its geometrical alignment depending on the isomeric state. It is the only band distinct in frequency between rhodopsin and isorhodopsin in the high-frequency region, suggesting that the specific interaction between the N-H and the chromophore contributes to the more efficient isomerization in rhodopsin than isorhodopsin. The stretching vibrations of the water O-H, cysteine S-H, and amide N-H of the peptide backbone decrease in frequency upon formation of bathorhodopsin, indicating that H-bonding around the chromophore becomes stronger in bathorhodopsin. This shows that at least a part of the energy absorbed in the chromophore is already transferred to the protein in bathorhodopsin by strengthened H-bonding. The chromophore-protein interaction as a suitable reaction field in rhodopsin is discussed on the basis of these observations.

Vibrationally Coherent Photochemistry in the Femtosecond Primary Event of Vision

Femtosecond pump-probe experiments reveal the impulsive production of photoproduct in the primary event in vision. The retinal chromophore of rhodopsin was excited with a 35-femtosecond pulse at 500 nanometers, and transient changes in absorption were measured with 10-femtosecond probe pulses. At probe wavelengths within the photo-product absorption band, oscillatory features with a period of 550 femtoseconds (60 wavenumbers) were observed whose phase and amplitude demonstrate that they are the result of nonstationary vibrational motion in the ground state of the photoproduct. The observation of coherent vibrational motion of the photoproduct supports the idea that the primary step in vision is a vibrationally coherent process and that the high quantum yield of the cis-->trans isomerization in rhodopsin is a consequence of the extreme speed of the excited-state torsional motion.

Rate of quantal transmitter release at the mammalian rod synapse

Under scotopic conditions, the mammalian rod encodes either one photon or none within its integration time. Consequently the signal presented to its synaptic terminal is binary. The synapse has a single active zone that releases neurotransmitter quanta tonically in darkness and pauses briefly in response to a rhodopsin isomerization by a photon. We asked: what minimum tonic rate would allow the postsynaptic bipolar cell to distinguish this pause from an extra-long interval between quanta due to the stochastic timing of release? The answer required a model of the circuit that included the rod convergence onto the bipolar cell and the bipolar cell's signal-to-noise ratio. Calculations from the model suggest that tonic release must be at least 40 quanta/s. This tonic rate is much higher than at conventional synapses where reliability is achieved by employing multiple active zones. The rod's synaptic mechanism makes efficient use of space, which in the retina is at a premium.

The First Step in Vision: Femtosecond Isomerization of Rhodopsin

The kinetics of the primary event in vision have been resolved with the use of femtosecond optical measurement techniques. The 11-cis retinal prosthetic group of rhodopsin is excited with a 35-femtosecond pump pulse at 500 nanometers, and the transient changes in absorption are measured between 450 and 580 nanometers with a 10-femtosecond probe pulse. Within 200 femtoseconds, an increased absorption is observed between 540 and 580 nanometers, indicating the formation of photoproduct on this time scale. These measurements demonstrate that the first step in vision, the 11-cis----11-trans torsional isomerization of the rhodopsin chromophore, is essentially complete in only 200 femtoseconds.

Mechanism of isomerization of rhodopsin studied by use of 11-cis-locked rhodopsin analogues excited with a picosecond laser pulse.

Primary photochemical behaviors of cattle rhodopsin analogues (Rh5 and Rh7) having cyclopenta- and cycloheptatrienylidene 11-cis-locked retinals (Ret5 and Ret7, respectively) were studied by excitation with a picosecond laser pulse (wavelength 532 nm; duration 21 ps). Picosecond absorption and fluorescence measurements of Rh5 showed formation of only a long-lived excited singlet state (tau l/e = 85 ps). The excited state of the retinal analogue having a five-membered ring was stabilized in protein (Rh5) more than in solvent (protonated Schiff base of Ret5; PSB5). Excitation of Rh7 produced two ground-state photoproducts, Rh7 (580) and Rh7 (630). According to the analysis of photon density dependency, Rh7 (580) was a single-photon product of Rh7, while Rh7 (630) was the photoproduct of Rh7 (580). Fluorescence emitted from a seven-membered ring system like Rh7 or a protonated Schiff base of Ret7 (PSB7) was weaker than that in a corresponding five-membered ring system, especially in protein (Rh7). The difference in photoreaction between Rh5 and Rh7 may originate from the difference in fixation of the 11-cis form. On the basis of the spectral and kinetic similarities between Rh7 (580) and photorhodopsin, a precursor of bathorhodopsin, it was proposed that both have twisted all-trans chromophores in the way of the isomerization. The protein moiety of rhodopsin which fixes the chromophore at both ends seems to accelerate the rotation of the C11-C12 double bond and to prevent it from going through relaxation processes other than the isomerization. This may be a plausible reason why rhodopsin has a large quantum yield (0.67).

Cyclic nucleotide levels of the photosensitive irises of Bufo marinus and Lophius

The sphincter pupillae muscle of several species contracts autonomously after light directly strikes the iris. The response is initiated by isomerization of rhodopsin in the sarcolemma. Light-induced release of CA2+ from internal stores is one step in the transduction process. The levels of cyclic AMP and cyclic GMP were measured after irises were stimulated by light. Stimulation by light does not produce measurable or consistent changes in the levels of either cyclic AMP or cyclic GMP within the photosensitive irises of at least two species, Bufo marinus and Lophius.

Changes in cGMP concentration correlate with some, but not all, aspects of the light-regulated conductance of frog rod photoreceptors.

Cyclic GMP has been implicated in controlling the light-regulated conductance of rod photoreceptors of the vertebrate retina. However, there is little direct evidence correlating changes in cGMP concentration with the light-regulated permeability mechanism in living cells. A preparation of intact frog rod outer segments suspended in a Ringer's medium containing low Ca2+ has been used to demonstrate that initial changes in total cellular cGMP concentration parallel changes in the light-regulated membrane current over a wide range of light intensities. At light intensities bleaching from 160 to 5.6 X 10(6) rhodopsin molecules/rod/s, decreases in the response latency for the cGMP kinetics parallel decreases in the latent period of the electrical response. Further, changes in the rate of the cGMP decrease parallel the rate of membrane current suppression as the light intensity is varied. Up to 10(5) cGMP molecules are hydrolyzed per photolyzed rhodopsin, consistent with in vitro studies showing that each bleached rhodopsin can activate over 100 phosphodiesterase molecules. Addition of the Ca2+ ionophore, A23187, does not affect the initial kinetics of the cGMP decrease or of the electrical response, excluding a direct role for Ca2+ in the initial events of phototransduction. These results are consistent with cGMP being the intracellular messenger that links rhodopsin isomerization with changes in membrane permeability upon illumination. It is unlikely, however, that light-induced changes in total cGMP concentration are the sole regulators of membrane current. This is suggested by several observations: at bright light intensities, the subsecond light-induced cGMP decrease is essentially complete prior to complete suppression of membrane current; maximal light-induced decreases in cGMP concentration occur at all light intensities tested, whereas the extent of membrane current suppression varies over the same range of light intensities; changing the external Ca2+ concentration from 1 mM to 10 nM in the dark causes an increase in membrane current that is significantly more rapid than corresponding changes in cGMP concentration. Thus, light-induced changes in total cellular cGMP concentration correlate with some, but not all, aspects of the visual excitation process in vertebrate photoreceptors.

Calcium ion, an intracellular messenger of light adaptation, also participates in excitation of Limulus photoreceptors.

Photoreceptor cells of Limulus ventral eyes were bathed in artificial sea water (ASW) that contained 10 mM-EGTA and no added Ca2+ (EGTA-ASW). Test flashes elicited responses that increased to a maximum size within 10 min in EGTA-ASW but did not change further when dark-adapted cells were bathed for an additional 35 min in this solution. Light responses progressively declined from this maximum size if the cells were repetitively illuminated in EGTA-ASW. In this state of reduced responsiveness, response amplitudes were further reduced by intracellular ionophoretic injection of EGTA; response amplitudes were increased by intracellular ionophoretic injection of Ca2+. Both of these findings are opposite to what is normally observed for cells bathed in ASW. Also, after repetitive illumination in EGTA-ASW, both the slope of the response versus intensity relationship became steeper and light responses often had a delayed increase in amplitude. The light responses and the response versus intensity relation returned to normal when the bathing medium was changed back to ASW containing 10 mM-Ca2+. The light-induced rise in luminescence recorded from photoreceptors injected with the photoprotein aequorin (the 'aequorin response') declined by at most 50% after dark-adapted photoreceptors were bathed in EGTA-ASW for 45 min. However, the aequorin response progressively declined by 98% if cells were repetitively illuminated while bathed in EGTA-ASW. The total intracellular Ca content of whole end-organs was measured by atomic absorption spectroscopy. Total intracellular Ca content did not change significantly while photoreceptors were bathed in EGTA-ASW even after repetitive illumination. We suggest that cytosolic Ca2+ is required by one or more steps in the mechanisms that link rhodopsin isomerization to both (i) an increase in the conductance of the cell membrane to Na+ and (ii) a release of Ca2+ from a light-labile store.

Fluorescence quantum yield of visual pigments: evidence for subpicosecond isomerization rates.

The fluorescence quantum yields (phi f) for bovine and squid rhodopsins are determined. Both pigments yield similar results, with an average value for phi f of 1.2 (+/- 0.5) X 10(-5). Since the estimated radiative lifetime of rhodopsin is 5 nsec, the rate constant of the process that competes with fluorescence must be on the order of 0.1 psec. Given the large quantum yield for isomerization of rhodopsin's retinal chromophore, this process is likely to correspond to the motion along retinal's C11-C12 torsional coordinate that leads to cis-trans isomerization. An empirical excited-state potential energy curve along this coordinate is derived. It is shown that subpicosecond torsional motion to highly twisted nonfluorescing regions of the potential is possible and, in fact, likely. Our results require the existence of a barrier-less excited-state potential energy curve and suggest that cis-trans isomerization occurs in less than 1 psec.

Chemical excitation of Limulus photoreceptors. I. Phosphatase inhibitors induce discrete-wave production in the dark.

Molybdate, tungstate, fluoride, vanadate, and GTP-gamma-S [guanosine-5'-0-(3-thiotriphosphate)] were injected into Limulus ventral photoreceptors by ionophoresis from microelectrodes. All of these drugs induce discrete waves of depolarization similar in waveform to, but smaller in amplitude than, those normally elicited by dim light. As for light-evoked waves, the amplitude of drug-induced waves decreases with light adaptation. For the compounds examined so far (fluoride, vanadate, GTP-gamma-S), the drug-induced waves share a reversal potential with light-induced discrete waves at about +15 mV. The induction of discrete waves by fluoride, vanadate, and molybdate was found to be reversible, whereas the induction of waves by GTP-gamma-S was not. Unlike fluoride and vanadate, which induce waves when added to the bath, molybdate appears to be ineffective when applied extracellularly. Because of the similarity of the drug-induced waves to light-induced discrete waves, we conclude that the drug-induced waves arise from a process similar or perhaps identical to visual excitation of the photoreceptor. However, the smaller size of drug-induced waves suggests that they arise at a stage of phototransduction subsequent to the isomerization of rhodopsin. On the basis of the chemical properties and action of the drugs, we suggest that discrete waves may arise through the activation of a GTP-binding protein.

Two components of electrical dark noise in toad retinal rod outer segments.

1. Physiological noise in the visual transduction mechanism was studied by recording membrane current from single rod outer segments in pieces of isolated toad retina. 2. The inward current in darkness showed spontaneous fluctuations which disappeared during the response to bright light. 3. The dark noise consisted of two components, a continuous fluctuation of rms amplitude about 0.2 pA and occasional discrete events about 1 pA in size. 4. Intervals between discrete events followed the exponential distribution expected of a Poisson process with a mean rate of about one event per 50 sec (20 degrees C). 5. The amplitude and power spectrum of the discrete events resembled those of single photon effects in the same rod, suggesting that discrete events may arise from spontaneous activation of single rhodopsin molecules. 6. The temperature dependence of the mean frequency of occurrence of discrete events gave an activation energy of 22 kcal mole-1, probably characteristic of thermal isomerization of rhodopsin. 7. The variance of the continuous component of the dark noise rose linearly with the length of the outer segment drawn into the suction electrode, indicating that this component is generated in the outer segment. 8. The power spectrum of a rod's continuous noise was usually fitted by the square of a Lorentzian with the same time constant as that of the four first-order delays in the cell's single photon response. The shot effects composing the continuous component thus appear to be shaped by two of four sequential processes in transduction. 9. The variance and spectrum of the continuous noise are interpreted to reflect shot effects about 1/400 the size of a single photon effect occurring at a frequency of 6 x 10(3) sec-1. 10. The rod's flash sensitivity was halved by a steady light to giving about 8 photoisomerizations sec-1. The much lower mean rate of discrete events indicates that Io in increment sensitivity experiments on individual receptors is not set by thermal activation of rhodopsin. 11. Values of sensitivity and time-to-peak flash response collected from many cells in darkness were correlated by the same power law relation obtaining in the presence of backgrounds. The correlation observed would be explained if a single variable controlled both the gain and time scale of several stages of the transduction mechanism in background light and in darkness.

Molecular dynamics of cis-trans isomerization in rhodopsin

Molecular dynamics of cis-trans isomerization in rhodopsin

The single-photon signal in rod bipolar cells of the dogfish retina.

1. Responses of depolarizing bipolar cells to dim light flashes were recorded with intracellular micro-electrodes in the dark-adapted retina of the dogfish, Scyliorhinus canicula. 2. Fluctuations in the responses were analysed by a method of matched filtering in order to improve the signal-to-noise ratio. 3. Both the mean and variance of the response amplitude increased linearly with light intensity for intensities not exceeding a mean of 1 photon absorbed per 50 rods. 4. On the assumption that the most significant source of the fluctuation is the quantal absorption of light by the rod outer segments, a single photoisomerization leads to a post-synaptic event of mean size 250 micronV. 5. The mean number of rods in the pool sending signals to a bipolar cell is estimated as 1600. Individual rod pools are 90-330 micrometer in diameter on the retinal surface. 6. It is estimated that the conductance of 1 divided by 400 of the total number of light-modulated ionic channels in the bipolar cell is increased by a single photon acting within its rod pool. 7. In the absence of a light stimulus, the residual noise in the output of a matched filter can be interpreted as due mainly to spontaneous isomerization of rhodopsin in the rods and behaves as the 'dark light' postulated to limit detection at absolute threshold.

Electrophysiological measurement of the number of rhodopsin molecules in single Limulus photoreceptors.

Two partly independent electrophysiological methods are described for measuring the number of rhodopsin molecules (R) in single ventral photoreceptors. Method 1 is based on measurements of the relative intensity required to elicit a quantal response and the relative intensity required to half-saturate the early receptor potential (ERP). Method 2 is based on measurements of the absolute intensity required to elicit a quantal response. Both methods give values of R approximately equal to 10(9). From these and other measurements, estimates are derived for the surface density of rhodopsin (8,000/micrometer2), the charge movement during the ERP per isomerized rhodopsin (20 X 10(-21) C), and the half-time for thermal isomerization of rhodopsin (36yr).

Rhodopsin photoproducts and rod sensitivity in the skate retina.

The late photoproducts that result from the isomerization of rhodopsin have been identified in the isolated all-rod retina of the skate by means of rapid spectrophotometry. The sequence in which these intermediates form and decay could be described by a scheme that incorporates two pathways for the degradation of metarhodopsin II (MII) to retinol: one via metarhodopsin III (MIII) and the other (which bypasses MIII) through retinal. Computer simulation of the model yielded rate constants and spectral absorbance coefficients for the late photoproducts which fit experimental data obtained at temperatures ranging from 7 degrees C to 27 degrees C. Comparing the kinetics of the thermal reactions with the changes in rod threshold that occur during dark adaptation indicated that the decay of MII and the fall in receptor thresholds exhibit similarities with regard to their temperature dependence. However, the addition of 2 mM hydroxylamine to a perfusate bathing the retina greatly accelerated the photochemical reactions, but had no significant effect on the rate of recovery of rod sensitivity. It appears, therefore, that the late bleaching intermediates do not control the sensitivities of skate rods during dark adaptation.

Visual pigments. V. Ground and excited-state acid dissociation constants of protonated all-trans retinal schiff base and correlation with theory.

Abstract— The acid dissociation constants of protonated all‐trans retinal Schiff base (SB‐H+) in a 50% water‐methanol solution at 0°C is 6–95 for the ground state and nominally 16–65 for the first excited singlet state, with a potential range of ˜ 12–21. These values are in qualitative agreement with the results of semiempirical MO calculations, which indicate that the total charge density on nitrogen is greater in the first excited singlet than in the ground state (QN* > QN). However, pertinent to vision, CNDO/2 calculations on all‐trans and 11‐cis Schiff base and SB‐H+ indicate that, for torsional angles of approximately 80–100° around the 11–12 double bond, QN* < QN. This result suggests that it may be possible for the proton to come off the imine nitrogen during isomerization from 11 ‐cis SB‐H+ to all‐trans SB‐H+. The potential consequence of this during isomerization of rhodopsin is the initiation of unfolding of the protein opsin.