STRUCTURE OF HYPSORHODOPSIN: ANALYSIS BY FOURIER TRANSFORM INFRARED SPECTROSCOPY AT 10 K

Abstract— Vibrational bands of hypsorhodopsin in the difference Fourier transform infrared spectra were identified as the bands which arose after formation of isorhodopsin by successive irradiations of bovine rhodopsin at 10 K with >500 nm light, and also as the bands disappeared upon conversion to bathorhodopsin by warming. The chromophore bands were assigned by the bands which shifted upon deuterium substitution of the polyene chain of the retinylidene chromophore. The presence of chromophore bands which shift by D₂O exchange clearly shows that the Schiff base chromophore of hypsorhodopsin is protonated. The amide I bands and several other protein bands of hypsorhodopsin appeared at the same frequencies as those of bathorhodopsin, but they are different from those of rhodopsin and isorhodopsin. Furthermore, like bathorhodopsin, hypsorhodopsin displays the C_l—H out-of-plane bending mode which is weakly coupled with C₁₂-_-–H out-of-plane mode. These facts show that hypsorhodopsin has a chromophore conformation and chromophore-opsin interaction more similar to bathorhodopsin than to rhodopsin and isorhodopsin.     

A resonance Raman study of octopus bathorhodopsin with deuterium labeled retinal chromophores.

The resonance Raman spectrum of octopus bathorhodopsin in the fingerprint region and in the ethylenic-Schiff base region have been obtained at 80 K using the "pump-probe" technique as have its deuterated chromophore analogues at the C7D; C8D; C8,C7D2; C10D; C11D; C11, C12D2; C14D; C15D; C14, C15D2; and N16D positions. While these data are not sufficient to make definitive band assignments, many tentative assignments can be made. Because of the close spectral similarity between the octopus bathorhodopsin spectrum and that of bovine bathorhodopsin, we conclude that the essential configuration of octopus bathorhodopsin's chromophore is all-trans like. The data suggest that the Schiff base, C = N, configuration is trans (anti). The observed conformationally sensitive fingerprint bands show pronounced isotope shifts upon chromophore deuteration. The size of the shifts differ, in certain cases, from those found for bovine bathorhodopsin. Thus, the internal mode composition of the fingerprint bands differs somewhat from bovine bathorhodopsin, suggesting a somewhat different in situ chromophore conformation. An analysis of the NH bend frequency, the Schiff base C = N stretch frequency, and its shift upon Schiff base deuteration suggests that the hydrogen bonding between the protonated Schiff base with its protein binding pocket is weaker in octopus bathorhodopsin than in bovine bathorhodopsin but stronger than that found in bacteriorhodopsin's bR568 pigment.     

SIMULATION ANALYSIS OF THE PHOTOCONVERSION PROCESS OF SQUID RHODOPSIN AT LIQUID HELIUM TEMPERATURE

The photoconversion process among squid rhodopsin, bathorhodopsin isorhodopsin and hypsorhodopsin was studied at liquid helium temperature. We evaluated the relative quantum yields of the photoconversion among four pigments by analysing the time-dependent change of absorption spectra. The result suggests that hypsorhodopsin is the common intermediate of rhodopsin and isorhodopsin, and there is no direct conversion between rhodopsin and isorhodopsin. Furthermore, rhodopsin converts to hypsorhodopsin or bathorhodopsin much more efficiently than does isorhodopsin, and bathorhodopsin does not convert directly to hypsorhodopsin. It was also found that rhodopsin and isorhodopsin convert to bathorhodopsin more efficiently than to hypsorhodopsin. In particular, the quantum yield of conversion from rhodopsin to bathorhodopsin was found to be about twice as large as that from rhodopsin to hypsorhodopsin. This result is somewhat in disagreement with the result obtained from the laser flash experiments at room and liquid nitrogen temperatures. The reason for the difference between the two experimental results is discussed.     

SQUID HYPSORHODOPSIN

Abstract— Squid hypsorhodopsin is produced by irradiating rhodopsin or isorhodopsin with yellow light (>480nm) at liquid He temperature (4K). Compared with cattle rhodopsin, squid rhodopsin easily converts to a photosteady state mixture composed of rhodopsin, isorhodopsin, hypsorhodopsin and bathorhodopsin at this temperature and the amount of hypsorhodopsin in the mixture is high. Hypsorhodopsin has a main absorption peak at 446 nm, and its extinction coefficient is 1.16 times larger than that of rhodopsin. On warming above 35 K, squid hypsorhodopsin converts to bathorhodopsin. A kinetic analysis indicates that the hypsorhodopsin can be formed not only from rhodopsin but also from isorhodopsin. On absorption of light. both squid bathorhodopsin and hypsorhodopsin convert to a mixture of rhodopsin and isorhodopsin.     

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.     

EXISTENCE OF HYPSORHODOPSIN AS THE FIRST INTERMEDIATE IN THE PRIMARY PHOTOCHEMICAL PROCESS OF CATTLE RHODOPSIN

It has long been believed that bathorhodopsin is the first intermediate of visual process for cattle rhodopsin. In the present paper hypsorhodopsin is shown to be the first intermediate by the use of picosecond spectropic technique. The main first intermediate, hypsorhodopsin, is formed with the time constant of 15 ± 5 ps. The time constant of the formation of bathorhodopsin from hypsorhodopsin is 50 ± 20 ps. Bathorhodopsin intermediates formed directly from excited state rhodopsin and those formed indirectly through hypsorhodopsin are 71/2^#% and 93%, respectively, of total bathorhodopsin intermediates in octylglucoside buffered solution. Batho intermediates formed directly and indirectly are 0% and 100%. respectively, of total batho intermediates in LDAO buffered solution.     

PICOSECOND LASER PHOTOLYSIS OF SQUID RHODOPSIN AT ROOM AND LOW TEMPERATURES

Abstract. Squid rhodopsin extracted with 2% digitonin (pH 10.5 or 7.0) was excited with a 347 nm light pulse from a mode-locked ruby laser at room temperature. Within 19 ps after the excitation, absorbance at 430 nm due to hypsorhodopsin increased and subsequently decreased with a decay time of 45 ± 10 ps. Absorbance at 550 nm due to bathorhodopsin increased with a rise time of 50 ± 10 ps. These results are the first observations of hypsorhodopsin at room temperature and clearly show that hypsorhodopsin is a precursor of bathorhodopsin which has been considered to be the earliest photoproduct in the photobleaching process of rhodopsin.
Hypsorhodopsin appeared with a rise time of 70 ± 10 ps at 421 nm at liquid nitrogen temperature without any bathorhodopsin being observed during the formation of hypsorhodopsin. An experiment using an N₂ laser showed that squid bathorhodopsin converted to lumirhodopsin with a decay time of about 300 ns at room temperature.     

THE ORIGIN OF THE RED-SHIFTED ABSORPTION MAXIMUM OF THE M412 INTERMEDIATE IN THE BACTERIORHODOPSIN PHOTOCYCLE

The factors that red shift the absorption maximum of the retinal Schiff base chromophore in the M₄₁₂ intermediate of bacteriorhodopsin photocycle relative to absorption in solution were investigated using a series of artificial pigments and studies of model compounds in solution. The artificial pigments derived from retinal analogs that perturb chromophore-protein interactions in the vicinity of the ring moiety indicate that a considerable part of the red shift may originate from interactions in the vicinity of the Schiff base linkage. Studies with model compounds revealed that hydrogen bonding to the Schiff base moiety can significantly red shift the absorption maximum. Furthermore, it was demonstrated that although s-trans ring-chain planarity prevails in the M₄₁₂ intermediate it does not contribute significantly (only ca 750 cm⁻¹) to the opsin shift observed in M₄₁₂. It is suggested that in M₄₁₂, the Schiff base linkage is hydrogen bonded to bound water and/or protein residues inducing a considerable red shift in the absorption maximum of the retinal chromophore.     

PROTON TRANSFER IN THE PRIMARY PROCESS OF VISION

Abstract— Proton transfer was theoretically examined as a possible primary process of vision. The motion of protons in the adiabatic potential of the Schiff base hydrogen bond was investigated in terms of quantum mechanics. The probability of proton transfer from the Schiff base nitrogen (i.e. the unprotonation of Schiff base) was found to increase as the retinal rotated around 11–12. double bond by 90°. The results also suggested that the proton transfer can take place before or during the transition from the excited to ground state (excited state proton transfer). We proposed that such excited state proton transfer is one of the elementary processes in primary visual photochemistry, and this process leads to the unprotonated visual pigment, hyposorhodopsin, which has been experimentally verified as one of the primary photoproducts of rhodopsin. The probability of this process could be comparable to the conventional process leading to the protonated intermediate, bathorhodopsin. The relation of these results with the recent experimental data is discussed.     

PICOSECOND AND NANOSECOND TSOMERIZATION KINETICS OF PROTONATED 11-CIS RETINYLIDENE SCHIFF BASES

Abstract— Picosecond and nonosecond spectroscopy has been used to study the isomerization mechanism of protonated 11- cis retinylidene Schiff bases. The formation and bleaching of absorption bands within 10 ps and corresponding decay and recovery within 11 ns indicate that the isomerization mechanism of the protonated Schiff bases is not identical to rhodopsin in which the primary photophysical event is probably due to electron transfer or partial isomerization of the chromophore to a nonplanar conformation.     

THE FORMATION OF TWO FORMS OF BATHORHODOPSIN AND THEIR OPTICAL PROPERTIES

Abstract— Using two kinds of rhodopsin preparations (digitonin extract and rod outer segments suspension), we measured changes in absorption spectra during the conversion of rhodopsin or isorhodopsin to a photosteady state mixture composed of rhodopsin, isorhodopsin and bathorhodopsin by irradiation with blue light (437 nm) at 77 K and during the reversion of bathorhodopsin to a mixture of rhodopsin and isorhodopsin by irradiation with red light (> 650 nm) at 77 K. The reaction kinetics could be expressed with only one exponential in the former case and with two exponentials in the latter case. These data suggest that both rhodopsin and isorhodopsin are composed of a single molecular species, while bathorhodopsin is composed of two molecular species, designated as bathorhodopsin₁ and bathorhodopsin₂. The absorption spectra of these bathorhodopsin were calculated by two different methods (kinetic method and warming-cooling method). The former was based on the kinetics of the conversion of two forms of bathorhodopsin by irradiation with the red light. The spectra obtained by this method were consistent with those obtained by the warming-cooling method. Bathorhodopsin₁ and bathorhodopsin₂ have Λ_max at 555 and 538 nm, respectively. The two forms of bathorhodopsin are interconvertible in the light, but not in the dark. Thus, we suggest that a rhodopsin molecule in the excited state relaxes to either bathorhodopsin₁ or bathorhodopsin₂ through one of the two parallel pathways.     

Absorption studies of neutral retinal Schiff base chromophores

The neutral retinal Schiff base is connected to opsin in UV sensing pigments and in the blue-shifted meta-II signaling state of the rhodopsin photocycle. We have designed and synthesized two model systems for this neutral chromophore and have measured their gas-phase absorption spectra in the electrostatic storage ring ELISA with a photofragmentation technique. By comparison to the absorption spectrum of the protonated retinal Schiff base in vacuo, we found that the blue shift caused by deprotonation of the Schiff base is more than 200 nm. The absorption properties of the UV absorbing proteins are thus largely determined by the intrinsic properties of the chromophore. The effect of approaching a positive charge to the Schiff base was also studied, as well as the susceptibility of the protonated and unprotonated chromophores to experience spectral shifts in different solvents.     

PRIMARY PHOTOCHEMISTRY OF BACTERIORHODOPSIN: COMPARISON OF FOURIER TRANSFORM INFRARED DIFFERENCE SPECTRA WITH RESONANCE RAMAN SPECTRA

Abstract —Fourier transform infrared (FTIR) difference spectra of the BR→rK transition in bacteriorhodopsin at 77→K are compared with analogous resonance Raman difference spectra obtained using a spinning sample cell at 77→K. The vibrational frequencies observed in the FTIR spectra of native purple membrane and of purple membrane regenerated with 15-deuterioretinal are in good agreement with the frequencies observed in the Raman spectra, indicating that the lines in the FTIR difference spectra arise predominantly from retinal chromophore vibrations. This agreement confirms that the spinning cell method for obtaining resonance Raman spectra of K minimizes potential contributions from unwanted photoproducts. The unexpected similarity between the resonance Raman scattering intensities and the FTIR absorption intensities for BR and K is discussed in terms of the delocalized electronic structure of the chromophore. Finally, comparison of the Schiff base regions of the K Raman and FTIR spectra provide additional information on the assignment of its Schiff base vibration.     

FEMTOSECOND STUDIES OF PRIMARY PHOTOPROCESSES IN OCTOPUS RHODOPSIN

Abstract— Femtosecond spectroscopy of octopus rhodopsin in H₂O and D₂O was performed over a very wide spectral region of 400–1000 nm. Transient gain and absorption from the excited state were observed for the first time around 650 and 700 nm, respectively, just after 300 fs pulse excitation. Bathorhodopsin was formed within 400 fs from the excited state; therefore, the cis-trans isomerization completes within 400 fs. The first intermediate "primerhodopsin" found in our previous paper is most likely "quasi-thermal" bathorhodopsin, in which the local thermalization of the chromophore is achieved. Then cooling down of the chromophore to the surrounding protein temperature takes place with 20 ± 10 ps along with blue-shifting of a spectrum of 10 ± 5 nm. In addition to these observations, a prominent gain in the region of > 850 nm was observed and decayed with 2–3 ps in H₂O. A similar time constant was estimated for a partial decay of an induced absorption around 600 nm. This process may be related with two forms of bathorhodopsin reported previously. In this scheme, two forms of bathorhodopsin are formed with time constants of about 400 fs and 2 ps. In the sample in D₂O, time constant of 3–4 ps was obtained for the slower process.     

All-trans-N-retinylidenetryptamine Schiff base in surfactant solubilized water pools in heptane—a fluorescence study

All-trns-N-retinylidenetryptamine Schiff base was incorporated into aerosol-OT (AOT, sodium bis(2-ethylhexyl)sulphosuccinate)/heptane reverse micelles. This micellar system was used as a model to study the retinal-tryptophan interactions in retinal proteins. The retinylidene Schiff base remains stable in the presence of reverse micelle-solubilized water pools. Partition coefficient and microviscosity measurements show that the Schiff base is located in the micellar interphase. The results are discussed in terms of the interaction between the retinylidene chromophore and the active site environment of rhodopsin and bacteriorhodopsin. In the present model, the quencher and emitting units are covalently attached, and are separated by two carbon spacer units. The fluorescence emission data obtained for the micelle-intercalated Schiff base chromophore are compared with the fluorescence of the native protein and intermediates in the photochemical cycle of bacteriofhodopsin. A comparison of the data obtained for tryptamine and the Schiff base with the results available for bacteriorhodopsin and bacterioopsin reveals that there is a large degree of quenching on intercalation of the retinylidene chromophore in the vicinity of the fluorophore. Evidence provided by this model suggests that energy transfer to retinal can occur from tryptophan residues located in the retinal pocket in the native protein. Thus the retinylidene unit can act as a quencher of the energy of tryptophan, the nature and extent of which may depend on the conformation and relative orientation of the protein-bound fluorophore.     

Interaction of chromophore, 11-<Emphasis Type="Italic">cis</Emphasis>-retinal,with amino acid residues of the visual pigment rhodopsin in the region of protonated Schiff base: A molecular dynamics study

A molecular dynamics study of the dark adapted visual pigment rhodopsin molecule was carried out. The interaction of the chromophore group, 11-cis-retinal, with the nearest amino acid residues in the chromophore center of the molecule, namely, in the region of the protonated Schiff base linkage, was analyzed. Most likely, the interaction of the CH=NH bond with the negatively charged amino acid residue Glu113 cannot be described as a simple electrostatic interaction of two oppositely charged groups. One can propose that not only Glu113 but also Glu181 and Ser186 are involved in stabilization of the protonated Schiff base linkage. Accord-ing to calculations, Glu181 interacts, as the counter-ion, with the Schiff base indirectly via Ser186. The intramolecular mechanisms of protonated Schiff base stabilization in rhodopsin are discussed. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 1, pp. 19–27, January, 2007.     

Rhodopsin deactivation is affected by mutations of Tyrl91

The 9-methyl group of 11-cis retinal is important in the efficient formation of the active conformation of rhodopsin, Meta II. Here, Tyrl91 rhodopsin mutants were generated because of its proximity to that methyl group in the dark structure. If photoactivation results in movement of the 9-methyl group toward Tyrl91, the steric interactions involved with activation and/or deactivation might not be as tightly coupled in mutant proteins with smaller amino acids at that position. Tyrl91 mutations have no effect on the dark pigment. However, after photobleaching, the lifetime of Meta II is shorter; Meta II decays quickly into two inactive species: (1) a Meta III or Meta III-like species and (2) opsin and free retinal. The Meta III-like fraction maintains the covalent Schiff base linkage of the chromophore much longer than the wild type. On the other hand, the fast chromophore release is similar to cone pigments. Taken together, the data suggest that the role of the 9-methyl group after photo-isomerization is not only to form Meta II efficiently, but also to maintain its active conformation and allow for the timely hydrolysis of the Schiff base. Perturbation of this interaction effects changes in the hydrolysis of the Schiff base and for the case of the Y191A mutation the folded structure of the protein after photobleaching.     

INTERMOLECULAR INTERACTIONS IN VISUAL PIGMENTS. THE HYDROGEN BOND IN VISLON

Model studies including quantum chemical calculations and the measurement of infrared and ultraviolet spectra are presented as contributions to the elucidation of the nature of the photochemical step of vision. The importance of the hydrogen bond in which the protonated nitrogen of the retinal Schiff base is involved is stressed as well as that of the perturbation of the β-ionone ring by negative groups. It is suggested that by combining these two perturbations the low excitation energy of rhodopsin can be obtained without actual protonation of the Schiff-base prior to photon absorption. The variation of rhodopsin's color from one species to another could also be related to this. Protonation could be a consequence of photonabsorption and the higher basicity of the excited state. This, in turn, leads to the suggestion that the protonated species is actually bathorhodopsin, not rhodopsin. Comments are made on the identity of the (ππ*) state which is involved.     

Comparative Analysis of GPCR Crystal Structures†

The phototransduction cascade is perhaps the best understood model system for G protein‐coupled receptor (GPCR) signaling. Phototransduction links the absorption of a single photon of light to a decrease in cytosolic cGMP. Depletion of the cGMP pool induces closure of cGMP‐gated cation channels resulting in the hyperpolarization of photoreceptor cells and consequently a neuronal response. Many biochemical and both low‐ and high‐resolution structural approaches have been utilized to increase our understanding of rhodopsin, the key molecule of this signaling cascade. Rhodopsin, a member of the GPCR or seven‐transmembrane spanning receptor superfamily, is composed of a chromophore, 11‐cis‐retinal that is covalently bound by a protonated Schiff base linkage to the apo‐protein opsin at Lys²⁹⁶ (in bovine opsin). Upon absorption of a photon, isomerization of the chromophore to an all‐trans‐retinylidene conformation induces changes in the rhodopsin structure, ultimately converting it from an inactive to an activated state. This state allows it to activate the heterotrimeric G protein, transducin, by triggering nucleotide exchange. To fully understand the structural and functional aspects of rhodopsin it is necessary to critically examine crystal structures of its different photointermediates. In this review we summarize recent progress on the structure and activation of rhodopsin in the context of other GPCR structures.     

COOPERATIVITY OF THE DEHYDRATION BLUE-SHIFT OF BACTERIORHODOPSIN*

Abstract– Dehydration of purple membrane (PM) causes a hlue-shift of the absorbance maximum from 570 nm to about 530 nm [Lazarev and Terpugov (1980) Biochim. Biophys. Acta 590 .324–338; Hildebrandt and Stockburger (1984) Biochemistry 23 ,5539–5548]. The absorbance spectra of PM dried in films at pH 0, 7 and 11 were measured at controlled relative humidities (RH). At pH 7, a blue-shift was observed similar to that previously reported. At pH 0(1M H₂SO₄) a reversible transition was observed from the “acid blue membrane” (maximum near 600 nm at 100% RH) to a blue-shifted dehydrated pigment (maximum near 578 nm at 50% RH), with isosbestic points at 592 and 710 nm. At pH 11 (NaOH) the absorbance maximum shifted to 530 nm, similar to the dehydrated form at pH 7. The fraction of hydrated chromophore, X_h, was calculated (assuming only two chromophore states, hydrated and dehydrated) as a function of humidity and pH. The resulting curve at pH 7 showed a steep decline in X_h below 20% RH. Near this hydration level, water clusters on protein surfaces break up, causing side-chain pK reversals. The Hill coefficient for the transition was about 2, indicating the minimum number of water molecules involved in a cooperative transition. The results suggest that as few as two water molecules are coordinated to the protonated retinal Schiff base of bacteriorhodopsin. A mechanism for the pH 7 dehydration blue-shift is proposed, involving a pK reversal of the protonated Schiff base and a nearby carboxyl side chain. At pH 0, a sharp decline in X_h occurs between 100 and 70% RH. Near this hydration level, complete protein surface coverage by a water monolayer occurs. The Hill coefficient is about 20, suggesting involvement of a large region of the surface.