Excited-state structure and isomerization dynamics of the retinal chromophore in rhodopsin from resonance Raman intensities.

Resonance Raman excitation profiles have been measured for the bovine visual pigment rhodopsin using excitation wavelengths ranging from 457.9 to 647.1 nm. A complete Franck-Condon analysis of the absorption spectrum and resonance Raman excitation profiles has been performed using an excited-state, time-dependent wavepacket propagation technique. This has enabled us to determine the change in geometry upon electronic excitation of rhodopsin's 11-cis-retinal protonated Schiff base chromophore along 25 normal coordinates. Intense low-frequency Raman lines are observed at 98, 135, 249, 336, and 461 cm-1 whose intensities provide quantitative, mode-specific information about the excited-state torsional deformations that lead to isomerization. The dominant contribution to the width of the absorption band in rhodopsin results from Franck-Condon progressions in the 1,549 cm-1 ethylenic normal mode. The lack of vibronic structure in the absorption spectrum is shown to be caused by extensive progressions in low-frequency torsional modes and a large homogeneous linewidth (170 cm-1 half-width) together with thermal population of low-frequency modes and inhomogeneous site distribution effects. The resonance Raman cross-sections of rhodopsin are unusually weak because the excited-state wavepacket moves rapidly (approximately 35 fs) and permanently away from the Franck-Condon geometry along skeletal stretching and torsional coordinates.     

Determination of retinal chromophore structure in bacteriorhodopsin with resonance Raman spectroscopy

The analysis of the vibrational spectrum of the retinal chromophore in bacteriorhodopsin with isotopic derivatives provides a powerful "structural dictionary" for the translation of vibrational frequencies and intensities into structural information. Of importance for the proton-pumping mechanism is the unambiguous determination of the configuration about the C13=C14 and C=N bonds, and the protonation state of the Schiff base nitrogen. Vibrational studies have shown that in light-adapted BR568 the Schiff base nitrogen is protonated and both the C13=C14 and C=N bonds are in a trans geometry. The formation of K625 involves the photochemical isomerization about only the C13=C14 bond which displaces the Schiff base proton into a different protein environment. Subsequent Schiff base deprotonation produces the M412 intermediate. Thermal reisomerization of the C13=C14 bond and reprotonation of the Schiff base occur in the M412------O640 transition, resetting the proton-pumping mechanism. The vibrational spectra can also be used to examine the conformation about the C--C single bonds. The frequency of the C14--C15 stretching vibration in BR568, K625, L550 and O640 argues that the C14--C15 conformation in these intermediates is s-trans. Conformational distortions of the chromophore have been identified in K625 and O640 through the observation of intense hydrogen out-of-plane wagging vibrations in the Raman spectra (see Fig. 2). These two intermediates are the direct products of chromophore isomerization. Thus it appears that following isomerization in a tight protein binding pocket, the chromophore cannot easily relax to a planar geometry. The analogous observation of intense hydrogen out-of-plane modes in the primary photoproduct in vision (Eyring et al., 1982) suggests that this may be a general phenomenon in protein-bound isomerizations. Future resonance Raman studies should provide even more details on how bacterio-opsin and retinal act in concert to produce an efficient light-energy convertor. Important unresolved questions involve the mechanism by which the protein catalyzes deprotonation of the L550 intermediate and the mechanism of the thermal conversion of M412 back to BR568. Also, it has been shown that under conditions of high ionic strength and/or low light intensity two protons are pumped per photocycle (Kuschmitz & Hess, 1981). How might this be accomplished?(ABSTRACT TRUNCATED AT 400 WORDS)     

Structure of the retinal chromophore in the hRL intermediate of halorhodopsin from resonance raman spectroscopy

Time-resolved resonance Raman spectra of the hRL intermediate of halorhodopsin have been obtained. The structurally sensitive fingerprint region of the hRL spectrum is very similar to that of bacteriorhodopsin's L550 intermediate, which is known to have a 13-cis configuration. This indicates that hRL contains a 13-cis chromophore and that an all-trans----13-cis isomerization occurs in the halorhodopsin photocycle. hRL exhibits a Schiff base stretching mode at 1644 cm-1, which shifts to 1620 cm-1 in D2O. This demonstrates that the Schiff base linkage to the protein is protonated. The insensitivity of the C-C stretching mode frequencies to N-deuteriation suggests that the Schiff base configuration is anti. The 24 cm-1 shift of the Schiff base mode in D2O indicates that the Schiff base proton in hRL has a stronger hydrogen-bonding interaction with the protein than does hR578.     

Resonance Raman spectroscopy of sensory rhodopsin II from Natronobacterium pharaonis

Sensory rhodopsin II (pSRII), the photophobic receptor from Natronobacterium pharaonis, has been studied by time-resolved resonance Raman (RR) spectroscopy using the rotating cell technique. Upon excitation with low laser power, the RR spectra largely reflect the parent state pSRII(500) whereas an increase of the laser power leads to a substantial accumulation of long-lived intermediates contributing to the RR spectra. All RR spectra could consistently be analysed in terms of four component spectra which were assigned to the parent state pSRII(500) and the long-lived intermediates M(400), N(485) and O(535) based on the correlation between the C = C stretching frequency and the absorption maximum. The parent state and the intermediates N(485) and O(535) exhibit a protonated Schiff base. The C = N stretching frequencies and the H/D isotopic shifts indicate strong hydrogen bonding interactions of the Schiff base in pSRII(500) and O(535) whereas these interactions are most likely very weak in N(485).     

Molecular flow resonance Raman effect from retinal and rhodopsin.

We have performed resonance enhanced Raman measurements of retinal isomers in solution (all-trans, 11-cis, 9-cis, and 13-cis) and cetyltrimethylammonium bromide (CTAB) detergent extracts of bovine rhodopsin near physiological temperatures (17 degrees C). In order to measure these photolabile systems, we have developed a general technique which allows Raman measurements of any photosensitive material. This technique involves imposing a molecular velocity transverse to the Raman exciting laser beam sufficient to ensure that any given molecule moves through the beam so that it has little probability of absorbing a photon. We have also measured the resonance Raman spectra of crystals of the same retinal isomers. The data show that each isomer has a distinct and characteristic Raman spectra and that the spectrum of 11-cis-retinal is quite similar but not identical with that of rhodopsin and similarly for 9-cis-retinal compared with isorhodopsin. In agreement with previous work, the Raman data demonstrate that retinal and opsin are joined by a protonated Schiff base. Due to the fact that the Raman spectra of 11-cis-retinal (solution) and rhodopsin show bands near 998 and 1018 cm(-1), a spectral region previously assigned to C-Me stretching motions, it is suggested that 11-cis-retinal in solution is compased of a mixture of 12-s-trans and 12-s-cis, and that the conformation of rhodopsin is (perhaps distorted) 12-s-trans.     

All-trans retinal constitutes the functional chromophore in Chlamydomonas rhodopsin

Orientation of the green alga Chlamydomonas in light (phototaxis and stop responses) is controlled by a visual system with a rhodopsin as the functional photoreceptor. Here, we present evidence that in Chlamydomonas wild-type cells all-trans retinal is the predominant isomer and that it is present in amounts similar to that of the rhodopsin itself.

The ability of different retinal isomers and analog compounds to restore photosensitivity in blind Chlamydomonas cells (strain CC2359) was tested by means of flash-induced light scattering transients or by measuring phototaxis in a taxigraph. All-trans retinal reconstitutes behavioral light responses within one minute, whereas cis-isomers require at least 50 × longer incubation times, suggesting that the retinal binding site is specific for all-trans retinal. Experiments with 13-demethyl(dm)-retinal and short-chained analogs reveal that only chromophores with a β-methyl group and at least three double bonds in conjugation with the aldehyde mediate function. Because neither 13-dm-retinal, nor 9,12-phenylretinal restores a functional rhodopsin, a trans/13-cis isomerisation seems to take place in the course of the activation mechanism. We conclude that with respect to its chromophore, Chlamydomonas rhodopsin bears a closer resemblence to bacterial rhodopsins than to visual rhodopsins of higher animals.

     

Halorhodopsin and sensory rhodopsin contain a C6-C7 s-trans retinal chromophore.

Halorhodopsin (HR) and sensory rhodopsin (SR) have been regenerated with retinal analogues that are covalently locked in the 6-s-cis or 6-s-trans conformations. Both pigments regenerate more completely with the locked 6-s-trans retinal and produce analogue pigments with absorption maxima (577 nm for HR and 592 nm for SR) nearly identical to those of the native pigments (577 and 587 nm). This indicates that HR and SR bind retinal in the 6-s-trans conformation. The opsin shift for the locked 6-s-trans analogue in HR is 1,200 cm-1 less than that for the native chromophore (5,400 cm-1). The opsin shift for the 6-s-trans analogue in SR is 1,100 cm-1 less than that for the native retinal (5,700 cm-1). This demonstrates that approximately 20% of the opsin shift in these pigments arises from a protein-induced change in the chromophore conformation from twisted 6-s-cis in solution to planar 6-s-trans in the protein. The reduced opsin shift observed for the locked 6-s-cis analogue pigments compared with the locked 6-s-trans pigments may be due to a positive electrostatic perturbation near C7.     

Picosecond dynamics of G-protein coupled receptor activation in rhodopsin from time-resolved UV resonance Raman spectroscopy

The protein response to retinal chromophore isomerization in the visual pigment rhodopsin is studied using picosecond time-resolved UV resonance Raman spectroscopy. High signal-to-noise Raman spectra are obtained using a 1 kHz Ti:Sapphire laser apparatus that provides <3 ps visible (466 nm) pump and UV (233 nm) probe pulses. When there is no time delay between the pump and probe events, tryptophan modes W18, W16, and W3 exhibit decreased Raman scattering intensity. At longer pump-probe time delays of +5 and +20 ps, both tryptophan (W18, W16, W3, and W1) and tyrosine (Y1 + 2xY16a, Y7a, Y8a) peak intensities drop by up to 3%. These intensity changes are attributed to decreased hydrophobicity in the microenvironment near at least one tryptophan and one tyrosine residue that likely arise from weakened interaction with the beta-ionone ring of the chromophore following cis-to-trans isomerization. Examination of the crystal structure suggests that W265 and Y268 are responsible for these signals. These UV Raman spectral changes are nearly identical to those observed for the rhodopsin-to-Meta I transition, implying that impulsively driven protein motion by the isomerizing chromophore during the 200 fs primary transition drives key structural changes that lead to protein activation.     

Low-temperature solid-state 13C NMR studies of the retinal chromophore in rhodopsin

Magic angle sample spinning (MASS) 13C NMR spectra have been obtained of bovine rhodopsin regenerated with retinal prosthetic groups isotopically enriched with 13C at C-5 and C-14. In order to observe the 13C retinal chromophore resonances, it was necessary to employ low temperatures (-15-----35 degrees C) to restrict rotational diffusion of the protein. The isotropic chemical shift and principal values of the chemical shift tensor of the 13C-5 label indicate that the retinal chromophore is in the twisted 6-s-cis conformation in rhodopsin, in contrast to the planar 6-s-trans conformation found in bacteriorhodopsin. The 13C-14 isotropic shift and shift tensor principal values show that the Schiff base C = N bond is anti. Furthermore, the 13C-14 chemical shift (121.2 ppm) is within the range of values (120-123 ppm) exhibited by protonated (C = N anti) Schiff base model compounds, indicating that the C = N linkage is protonated. Our results are discussed with regard to the mechanism of wavelength regulation in rhodopsin.     

Resonance Raman spectroscopy of chemically modified retinals: Assigning the carbon-methyl vibrations in the resonance Raman spectrum of rhodopsin

To assign the observed vibrationsl modes in the resonance Raman spectrum of the retinylidene chromophore of rhodopsin, we have studied chemically modified retinals. The series of analogs investigated are the n-butyl retinals substituted at C₉ and C₁₃. The results obtained for the 11-cis isomer have clearly assigned the CCH₃ vibrational frequencies observed in the spectrum of the retinylidene chromophore. The data show that the C₍₉₎CH₃ stretching vibration can be assigned to the vibrational mode observed in the 1017 cm^?1 region, and the vibration detected at 997 cm^?1 can be assigned to the C₍₁₃CH₃ vibration. The C₍₅₎CH₃ stretching mode does not contribute to the vibrations observed in this region. The splitting in the C_(n)CH₃ (n = 9, 13) vibration is characteristic of the 11-cis conformation. The results on the modified retinals do not support the hypothesis that the splitting arises from equilibrium mixtures of 11-cis, 12-s-cis and 11-cis, 12-s-trans in solution. Thus, this splitting cannot be used to determine whether the chromophore in rhodopsin is in a 12-s-cis or 12-s-trans conformation. However, our results demonstrate that there are other vibrational modes in the spectra which are sensitive to this conformational equilibrium and we use the presence of a strong ~ 1271 cm^?1 mode in bovine and squid rhodopsin spectra as an indication that the chromophore in these pigments is 11-cis, 12-s-trans.     

Spectral tuning in sensory rhodopsin I from Salinibacter ruber

Organisms utilize light as energy sources and as signals. Rhodopsins, which have seven transmembrane α-helices with retinal covalently linked to a conserved Lys residue, are found in various organisms as distant in evolution as bacteria, archaea, and eukarya. One of the most notable properties of rhodopsin molecules is the large variation in their absorption spectrum. Sensory rhodopsin I (SRI) and sensory rhodopsin II (SRII) function as photosensors and have similar properties (retinal composition, photocycle, structure, and function) except for their λ(max) (SRI, ～560 nm; SRII, ～500 nm). An expression system utilizing Escherichia coli and the high protein stability of a newly found SRI-like protein, SrSRI, enables studies of mutant proteins. To determine the residue contributing to the spectral shift from SRI to SRII, we constructed various SRI mutants, in which individual residues were substituted with the corresponding residues of SRII. Three such mutants of SrSRI showed a large spectral blue-shift (>14 nm) without a large alteration of their retinal composition. Two of them, A136Y and A200T, are newly discovered color tuning residues. In the triple mutant, the λ(max) was 525 nm. The inverse mutation of SRII (F134H/Y139A/T204A) generated a spectral-shifted SRII toward longer wavelengths, although the effect is smaller than in the case of SRI, which is probably due to the lack of anion binding in the SRII mutant. Thus, half of the spectral shift from SRI to SRII could be explained by only those three residues taking into account the effect of Cl(-) binding.     

Effects of modifications of the retinal beta-ionone ring on archaebacterial sensory rhodopsin I.

Ring desmethyl and acyclic analogues of all-trans retinal were incorporated into the apoprotein of the phototaxis receptor sensory rhodopsin I (SR-I) in Halobacterium halobium membranes. All modified retinals generate SR-I analogue pigments which exhibit "opsin shifts," i.e., their absorption spectra are shifted to longer wavelengths compared with model protonated Schiff bases of the same analogues. Each SR-I pigment analogue exhibits cyclic photochemical reactions as monitored by flash spectroscopy, but the analogue photocycles differ from that of native SR-I by exhibiting pronounced biphasic recovery of flash-induced absorption changes and abnormal flash-induced absorption difference spectra. Despite perturbations in the photochemical properties, the SR-I pigment analogues are capable of both attractant (single photon) and repellent (two photon) phototaxis signaling in cells. Our interpretation is that the hydrophobic ring substituents interact with the binding pocket to maintain the correct configuration for native SR-I absorption and photochemistry, but these interactions are not essential for the physiological function of SR-I as a dual attractant/repellent phototaxis receptor. These results support the conclusion emerging from several studies that the photoactivation process that triggers the conformation changes of SR-I and the related proton pump bacteriorhodopsin is conserved despite the different biological functions of their photoactivation.     

Structure of the retinal chromophore in 7,9-dicis-rhodopsin

Bovine rhodopsin was bleached and regenerated with 7,9-dicis-retinal to form 7,9-dicis-rhodopsin, which was purified on a concanavalin A affinity column. The absorption maximum of the 7,9-dicis pigment is 453 nm, giving an opsin shift of 1600 cm-1 compared to 2500 cm-1 for 11-cis-rhodopsin and 2400 cm-1 for 9-cis-rhodopsin. Rapid-flow resonance Raman spectra have been obtained of 7,9-dicis-rhodopsin in H2O and D2O at room temperature. The shift of the 1654-cm-1 C = N stretch to 1627 cm-1 in D2O demonstrates that the Schiff base nitrogen is protonated. The absence of any shift in the 1201-cm-1 mode, which is assigned as the C14-C15 stretch, or of any other C-C stretching modes in D2O indicates that the Schiff base C = N configuration is trans (anti). Assuming that the cyclohexenyl ring binds with the same orientation in 7,9-dicis-, 9-cis-, and 11-cis-rhodopsins, the presence of two cis bonds requires that the N-H bond of the 7,9-dicis chromophore points in the opposite direction from that in the 9-cis or 11-cis pigment. However, the Schiff base C = NH+ stretching frequency and its D2O shift in 7,9-dicis-rhodopsin are very similar to those in 11-cis- and 9-cis-rhodopsin, indicating that the Schiff base electrostatic/hydrogen-bonding environments are effectively the same. The C = N trans (anti) Schiff base geometry of 7,9-dicis-rhodopsin and the insensitivity of its Schiff base vibrational properties to orientation are rationalized by examining the binding site specificity with molecular modeling.     

The retinal structure of channelrhodopsin-2 assessed by resonance Raman spectroscopy

Channelrhodopsin-2 mediates phototaxis in green algae by acting as a light-gated cation channel. As a result of this property, it is used as a novel optogenetic tool in neurophysiological applications. Structural information is still scant and we present here the first resonance Raman spectra of channelrhodopsin-2. Spectra of detergent solubilized and lipid-reconstituted protein were recorded under pre-resonant conditions to exclusively probe retinal in its electronic ground state. All-trans retinal was identified to be the favoured configuration of the chromophore but significant contributions of 13-cis were detected. Pre-illumination hardly changed the isomeric composition but small amounts of presumably 9-cis retinal were found in the light-adapted state. Spectral analysis suggested that the Schiff base proton is strongly hydrogen-bonded to a nearby water molecule.     

Transverse location of the retinal chromophore of rhodopsin in rod outer segment disc membranes

Diffusion-enhanced fluorescence energy transfer was used to study the structure of photoreceptor membranes from bovine retinal rod outer segments. The fluorescent energy donor was Tb³⁺ chelated to dipicolinate and the acceptor was the 11-cis retinal chromophore of rhodopsin in vesicles made from disc membranes. The rapid-diffusion limit for energy transfer was attained in these experiments because of the long excited state lifetime of the terbium donor (~2 ms). Under these conditions, energy transfer is very sensitive to a, the distance of closest approach between the donor and acceptor (Thomas et al., 1978). Vesicles containing terbium dipicolinate in their inner aqueous space were prepared by sonicating disc membranes in the presence of this chelate and chromatographing this mixture on a gel filtration column. The sidedness of rhodopsin in these vesicles was the same as in native disc membranes. The transfer efficiency from terbium to retinal in this sample was 43%. For an R₀ value of 46.7 Å and an average vesicle diameter of 650 Å, this corresponds to an a value of 22 Å from the inner aqueous space of the vesicle. The distance of closest approach from the external aqueous space, determined by adding terbium dipicolinate to a suspension of already formed vesicles, was found to be 28 Å. These values of a show that the retinal chromophore is far from both aqueous surfaces of the disc membrane. Hence, the transverse location of the retinal chromophore is near the center of the hydrophobic core of the disc membrane. These findings suggest that conformational changes induced by photoisomerization are transmitted through a distance of at least 20 Å within rhodopsin to trigger subsequent events in visual excitation.     

FT-IR difference spectroscopy elucidates crucial interactions of sensory rhodopsin I with the cognate transducer HtrI

The phototaxis receptor sensory rhodopsin I (SRI) from Halobacterium salinarum interacts with its cognate transducer (HtrI) forming a transmembrane complex. After light excitation of the chromophore all-trans retinal, SRI undergoes structural changes that are ultimately transmitted to HtrI. The interaction of SRI with HtrI results in the closure of the receptor's proton pathway, which renders the photocycle recovery kinetics of SRI pH-independent. We demonstrate on heterologously expressed and reconstituted SRI-HtrI fusion proteins that the transmembrane part of HtrI (residues 1-52) as well as the downstream cytoplasmic part (residues 53-147) exhibit conformational changes after light excitation. The sum of these conformational changes is similar to those observed in the fusion constructs SRI-HtrI 1-71 and SRI-HtrI 1-147, which display pH-independent receptor kinetics. These results indicate the occurrence of spatially distinct conformational changes that are required for functional signal transmission. Kinetic and spectroscopic analysis of HtrI point mutants of Asn53 provides evidence that this residue is involved in the receptor-transducer interaction. We suggest that Asn53 plays a role similar to that of Asn74 of the HtrII from Natronobacterium pharaonis, the latter forming a hydrogen bond to the receptor within the membrane.     

Structural analysis and dynamics of retinal chromophore in dark and meta I states of rhodopsin from 2H NMR of aligned membranes

Rhodopsin is a prototype for G protein-coupled receptors (GPCRs) that are implicated in many biological responses in humans. A site-directed (2)H NMR approach was used for structural analysis of retinal within its binding cavity in the dark and pre-activated meta I states. Retinal was labeled with (2)H at the C5, C9, or C13 methyl groups by total synthesis, and was used to regenerate the opsin apoprotein. Solid-state (2)H NMR spectra were acquired for aligned membranes in the low-temperature lipid gel phase versus the tilt angle to the magnetic field. Data reduction assumed a static uniaxial distribution, and gave the retinylidene methyl bond orientations plus the alignment disorder (mosaic spread). The dark-state (2)H NMR structure of 11-cis-retinal shows torsional twisting of the polyene chain and the beta-ionone ring. The ligand undergoes restricted motion, as evinced by order parameters of approximately 0.9 for the spinning C-C(2)H(3) groups, with off-axial fluctuations of approximately 15 degrees . Retinal is accommodated within the rhodopsin binding pocket with a negative pre-twist about the C11=C12 double bond that explains its rapid photochemistry and the trajectory of 11-cis to trans isomerization. In the cryo-trapped meta I state, the (2)H NMR structure shows a reduction of the polyene strain, while torsional twisting of the beta-ionone ring is maintained. Distortion of the retinal conformation is interpreted through substituent control of receptor activation. Steric hindrance between trans retinal and Trp265 can trigger formation of the subsequent activated meta II state. Our results are pertinent to quantum and molecular mechanics simulations of ligands bound to GPCRs, and illustrate how (2)H NMR can be applied to study their biological mechanisms of action.