Relative orientation between the beta-ionone ring and the polyene chain for the chromophore of rhodopsin in native membranes

Rotational resonance solid state nuclear magnetic resonance has been used to determine the relative orientation of the beta-ionone ring and the polyene chain of the chromophore 11-Z-retinylidene of rhodopsin in rod outer segment membranes from bovine retina. The bleached protein was regenerated with either 11-Z-[8,18-(13)C(2)]retinal or 11-Z-[8,16/17(13)C(2)]retinal, the latter having only one (13)C label at either of the chemically equivalent positions 16 and 17. Observation of (13)C selectively enriched in the ring methyl groups, C16/17, revealed alternative conformational states for the ring. Minor spectral components comprised around 26% of the chromophore. The major conformation (approximately 74%) has the chemical shift resolution required for measuring internuclear distances to (13)C in the retinal chain (C8) separately from each of these methyl groups. The resulting distance constraints, C8 to C16 and C17 (4.05 +/- 0.25 A) and from C8 to C18 (2.95 +/- 0.15 A), show that the major portion of retinylidene in rhodopsin has a twisted 6-s-cis conformation. The more precise distance measurement made here between C8 and C18 (2.95 A) predicts that the chain is twisted out-of-plane with respect to the ring by a modest amount (C5-C6-C7-C8 torsion angle = -28 +/- 7 degrees ).     

10,20-Methanorhodopsins: (7E,9E,13E)-10,20-methanorhodopsin and (7E,9Z,13Z)-10,20-methanorhodopsin. 11-cis-locked rhodopsin analog pigments with unusual thermal and photo-stability

Synthesis of the retinal analog, 10,20-methanoretinal (R6), where the 11Z conformation is locked in a six-membered ring, yielded four stereoisomers (7E,9E,13E, 7E,9E,13Z, 7E,9Z,13E and 7E,9Z,13Z). These four isomers were separated by straight-phase isocratic HPLC and identified by 1H-NMR and NOE analysis. All isomers smoothly recombined with bovine opsin at a relatively high rate (5-10% of that of the natural chromophore 11Z-retinal). The corresponding 13E and 13Z isomers yielded identical analog pigments, probably due to rapid thermal isomerization around the C13 = C14 double bond. The (7E,9E)-isomers produced a pigment with maximal absorbance at 510 nm, while the pigment produced from the (7E,9Z)-isomers had maximal absorbance at 494 nm. Based upon kinetic considerations, the chromophore structure in the 510-nm-absorbing pigment should be (7E,9E,13E), i.e. equivalent to 11Z-retinal and rhodopsin, while the chromophore structure in the 494-nm-absorbing pigment should be (7E,9Z,13Z), i.e. equivalent to (9Z,11Z,13Z)-rhodopsin, an isorhodopsin analog. In analogy to the 11-cis-locked rhodopsin analogs Rh5 and Rh7, the 510-nm-absorbing pigment, (7E,9E,13E)-10,20-methanorhodopsin, was dubbed Rh6 and the 494-nm-absorbing pigment. (7E,9Z,13Z)-10,20-methanorhodopsin, was dubbed Iso6. The opsin shift of Rh6 (2660 cm-1) is practically identical to that of rhodopsin itself (2650 cm-1). Rh6 and Iso6 are nearly as stable as rhodopsin towards hydroxylamine and solubilization in detergent solution and could be easily purified and reconstituted into proteoliposomes by established procedures. Due to the 11-cis-lock, Rh6 is much less photolabile (bleaching rate less than 1%) than rhodopsin, but it is not completely photostable, probably since photoisomerization around the C7 = C8, C9 = C10 and C13 = C14 bonds is allowed. Illumination of either Rh6 or Iso6 does not generate the common photointermediates but instead produces a complex pattern of photochemical transitions, which during continuous illumination leads to the same final steady state, absorbing at 498 nm. This process is accompanied by a slow but steady loss of pigment, probably due to hydrolytic release of chromophore, which is markedly accelerated in the presence of hydroxylamine. In a physiological assay (light-dependent activation of rod cGMP phosphodiesterase) Rh6 is only marginally active and this probably reflects conformational changes accompanying the above-mentioned photochemical transitions. This supports the concept that normal rhodopsin-based phototransduction requires 11Z to all-E isomerization.(ABSTRACT TRUNCATED AT 400 WORDS)     

Conformation and orientation of the retinyl chromophore in rhodopsin: a critical evaluation of recent NMR data on the basis of theoretical calculations results in a minimum energy structure consistent with all experimental data

In the absence of a high-resolution diffraction structure, the orientation and conformation of the protonated Schiffs base retinylidinium chromophore of rhodopsin within the opsin matrix has been the subject of much speculation. There have been two recent reliable and precise NMR results that bear on this issue. One involves a determination of the C20-C10 and C20-C11 distances by Verdegem et al. [Biochemistry 38, 11316-11324 (1999)]. The other is the determination of the orientation of the methine C to methyl group vectors C5-C18, C9-C19, and C13-C20 relative to the membrane normal by Gr?bner et al. [Nature 405 (6788), 810-813 (2000)]. Using molecular orbital methods that include extensive configuration interaction, we have determined what we propose to be the minimum energy conformation of this chromophore. The above NMR results permit us to check this structure in the C10-C11=C12-C13 region and then to check the global structure via the relative orientation of the three C18, C19, and C20 methyl groups. This method provides a detailed structure and also the orientation for the retinyl chromophore relative to the membrane normal and argues strongly that the protein does not appreciably alter the chromophore geometry from its minimum energy configuration that is nearly planar s-trans at the 6-7 bond. Finally, the chromophore structure and orientation presented in the recently published X-ray diffraction structure is compared with our proposed structure and with the deuterium NMR results.     

Origin and consequences of steric strain in the rhodopsin binding pocket

To study the origin and the effects of steric strain on the chromophore conformation in rhodopsin, we have performed quantum-mechanical calculations on the wild-type retinal chromophore and four retinal derivatives, 13-demethyl-, 10-methyl-13-demethyl-, 10-methyl-, and 9-demethylretinal. For the dynamics of the whole protein, a combined quantum mechanics/molecular mechanics method (DFTB/CHARMM) was used and for the calculation of excited-state properties the nonempirical CASSCF/CASPT2 method. After relaxation inside the protein, all chromophores show significant nonplanar distortions from C10 to C13, most strongly for 10-methylretinal and least pronounced for 9-demethylretinal. In all five cases, the dihedral angle of the C10-C11=C12-C13 bond is negative which attests to the strong chiral discrimination exerted by the protein pocket. The calculations show that the nonplanar distortion of the chromophore, including the sense of rotation, is caused by a combination of two effects: the fitting of both ends to the protein matrix which imposes a distance constraint and the bonding arrangement at the Schiff base terminus. With both the counterion Glu113 and Lys296 displaced off the plane of the chromophore, their binding to N16 exerts a torque on the chromophore. As a result, the polyene chain, from N16 to C13, is twisted in a clockwise manner against the remaining part of the chromophore, leading to a C11=C12 bond with the observed negative dihedral angle. Shifts of the absorption maxima are reproduced correctly, in particular, the red shift of the 10-methyl and the strong blue shift of the 9-demethyl analogue relative to the wild type. Calculated positive rotatory strengths of the alpha-CD bands are in agreement with the calculated absolute conformation of the mutant chromophores.     

Regeneration of bovine and octopus opsins in situ with natural and artificial retinals

We consider the problem of color regulation in visual pigments for both bovine rhodopsin (lambda max = 500 nm) and octopus rhodopsin (lambda max = 475 nm). Both pigments have 11-cis-retinal (lambda max = 379 nm, in ethanol) as their chromophore. These rhodopsins were bleached in their native membranes, and the opsins were regenerated with natural and artificial chromophores. Both bovine and octopus opsins were regenerated with the 9-cis- and 11-cis-retinal isomers, but the octopus opsin was additionally regenerated with the 13-cis and all-trans isomers. Titration of the octopus opsin with 11-cis-retinal gave an extinction coefficient for octopus rhodopsin of 27,000 +/- 3000 M-1 cm-1 at 475 nm. The absorption maxima of bovine artificial pigments formed by regenerating opsin with the 11-cis dihydro series of chromophores support a color regulation model for bovine rhodopsin in which the chromophore-binding site of the protein has two negative charges: one directly hydrogen bonded to the Schiff base nitrogen and another near carbon-13. Formation of octopus artificial pigments with both all-trans and 11-cis dihydro chromophores leads to a similar model for octopus rhodopsin and metarhodopsin: there are two negative charges in the chromophore-binding site, one directly hydrogen bonded to the Schiff base nitrogen and a second near carbon-13. The interaction of this second charge with the chromophore in octopus rhodopsin is weaker than in bovine, while in metarhodopsin it is as strong as in bovine.     

Light-stable rhodopsin. I. A rhodopsin analog reconstituted with a nonisomerizable 11-cis retinal derivative.

With the aim of preparing a light-stable rhodopsin-like pigment, an analog, II, of 11-cis retinal was synthesized in which isomerization of the C11-C12 cis-double bond is blocked by a cyclohexene ring built around the C10 to C13-methyl. The analog II formed a rhodopsin-like pigment (rhodopsin-II) with opsin expressed in COS-1 cells and with opsin from rod outer segments. The rate of rhodopsin-II formation from II and opsin was approximately 10 times slower than that of rhodopsin from 11-cis retinal and opsin. After solubilization in dodecyl maltoside and immunoaffinity purification, rhodopsin-II displayed an absorbance ratio (A280nm/A512nm) of 1.6, virtually identical with that of rhodopsin. Acid denaturation of rhodopsin-II formed a chromophore with lambda max, 452 nm, characteristic of protonated retinyl Schiff base. The ground state properties of rhodopsin-II were similar to those of rhodopsin in extinction coefficient (41,200 M-1 cm-1) and opsin-shift (2600 cm-1). Rhodopsin-II was stable to hydroxylamine in the dark, while light-dependent bleaching by hydroxylamine was slowed by approximately 2 orders of magnitude relative to rhodopsin. Illumination of rhodopsin-II for 10 s caused approximately 3 nm blue-shift and 3% loss of visible absorbance. Prolonged illumination caused a maximal blue-shift up to approximately 20 nm and approximately 40% loss of visible absorbance. An apparent photochemical steady state was reached after 12 min of illumination. Subsequent acid denaturation indicated that the retinyl Schiff base linkage was intact. A red-shift (approximately 12 nm) in lambda max and a 45% recovery of visible absorbance was observed after returning the 12-min illuminated pigment to darkness. Rhodopsin-II showed marginal light-dependent transducin activation and phosphorylation by rhodopsin kinase.     

Light-stable rhodopsin. II. An opsin mutant (TRP-265----Phe) and a retinal analog with a nonisomerizable 11-cis configuration form a photostable chromophore.

In order to prepare a completely light-stable rhodopsin, we have synthesized an analog, II, of 11-cis retinal in which isomerization at the C11-C12 cis-double bond is blocked by formation of a cyclohexene ring from the C10 to C13-methyl. We used this analog to generate a rhodopsin-like pigment from opsin expressed in COS-1 cells and opsin from rod outer segments (Bhattacharya, S., Ridge, K.D., Knox, B.E., and Khorana, H. G. (1992) J. Biol. Chem. 267, 6763-6769). The pigment (lambda max, 512 nm) formed from opsin and analog II (rhodospin-II) showed ground state properties very similar to those of rhodopsin, but was not entirely stable to light. In the present work, 12 opsin mutants (Ala-117----Phe, Glu-122----Gln(Ala, Asp), Trp-126----Phe(Leu, Ala), Trp-265----Ala(Tyr, Phe), Tyr-268----Phe, and Ala-292----Asp), where the mutations were presumed to be in the retinal binding pocket, were reconstituted with analog II. While all mutants formed rhodopsin-like pigments with II, blue-shifted (12-30 nm) chromophores were obtained with Ala-117----Phe, Glu-122----Gln(Ala), Trp-126----Leu(Ala), and Trp-265----Ala(Tyr, Phe) opsins. The extent of chromophore formation was markedly reduced in the mutants Ala-117----Phe and Trp-126----Ala. Upon illumination, the reconstituted pigments showed varying degrees of light sensitivity; the mutants Trp-126----Phe(Leu) showed light sensitivity similar to wild-type. Continuous illumination of the mutants Glu-122----Asp, Trp-265----Ala, Tyr-268----Phe, and Ala-292----Asp resulted in hydrolysis of the retinyl Schiff base. Markedly reduced light sensitivity was observed with the mutant Trp-265----Tyr, while the mutant Trp-265----Phe was light-insensitive. Consistent with this result, the mutant Trp-265----Phe showed no detectable light-dependent activation of transducin or phosphorylation by rhodopsin kinase.     

Ultra-high-field MAS NMR assay of a multispin labeled ligand bound to its G-protein receptor target in the natural membrane environment: electronic structure of the retinylidene chromophore in rhodopsin

11-Z-[8,9,10,11,12,13,14,15,19,20-(13)C10]Retinal prepared by total synthesis is reconstituted with opsin to form rhodopsin in the natural lipid membrane environment. The 13C shifts are assigned with magic angle spinning NMR dipolar correlation spectroscopy in a single experiment and compared with data of singly labeled retinylidene ligands in detergent-solubilized rhodopsin. The use of multispin labeling in combination with 2-D correlation spectroscopy improves the relative accuracy of the shift measurements. We have used the chemical shift data to analyze the electronic structure of the retinylidene ligand at three levels of understanding: (i) by specifying interactions between the 13C-labeled ligand and the G-protein-coupled receptor target, (ii) by making a charge assessment of the protonation of the Schiff base in rhodopsin, and (iii) by evaluating the total charge on the carbons of the retinylidene chromophore. In this way it is shown that a conjugation defect is the predominant ground-state property governing the molecular electronics of the retinylidene chromophore in rhodopsin. The cumulative chemical shifts at the odd-numbered carbons (Delta(sigma)odd) of 11-Z-protonated Schiff base models relative to the unprotonated Schiff base can be used to measure the extent of delocalization of positive charge into the polyene. For a series of 11-Z-protonated Schiff base models and rhodopsin, Delta(sigma)odd appears to correlate linearly with the frequency of maximum visible absorption. Since rhodopsin has the largest value of Delta(sigma)odd, the data contribute to existing and converging spectroscopic evidence for a complex counterion stabilizing the protonated Schiff base in the binding pocket.     

Determination of a molecular torsional angle in the metarhodopsin-I photointermediate of rhodopsin by double-quantum solid-state NMR

We present a solid-state NMR study of metarhodopsin-I, the pre-discharge intermediate of the photochemical signal transduction cascade of rhodopsin, which is the 41 kDa integral membrane protein that triggers phototransduction in vertebrate rod cells. The H-C10-C11-H torsional angles of the retinylidene chromophore in bovine rhodopsin and metarhodopsin-I were determined simultaneously in the photo-activated membrane-bound state, using double-quantum heteronuclear local field spectroscopy. The torsional angles were estimated to be || = 160 ± 10° for rhodopsin and = 180 ± 25° for metarhodopsin-I. The result is consistent with current models of the photo-induced conformational transitions in the chromophore, in which the 11-Z retinal ground state is twisted, while the later photointermediates have a planar all-E conformation.     

Studies on structure and function of rhodopsin by use of cyclopentatrienylidene 11-cis-locked-rhodopsin

The photochemical reaction of cyclopentatrienylidene 11-cis-locked-rhodopsin derived from cyclopentatrienylidene 11-cis-locked-retinal and cattle opsin was spectrophotometrically studied. The difference absorption spectrum between the cyclopentatrienylidene 11-cis-locked-rhodopsin and its retinal oxime had its maximum at 495 nm (P-495). Irradiation of P-495 at -196 degrees C with either blue light or orange light caused no spectral change, supporting the cis-trans isomerization hypothesis for formation of bathorhodopsin. Upon irradiation of P-495 at 0 degree C with orange light, however, its absorption spectrum shifted to a shorter wavelength owing to formation of a hypsochromic product. The difference absorption spectrum between this product (P-466) and its retinal oxime showed its maximum at 466 nm. Analysis of retinal isomers by high-performance liquid chromatography showed that this spectral shift was not accompanied by photoisomerization of the chromophore. P-466 could almost completely be photoconverted to the original pigment (P-495) by irradiation at 0 degree C with blue light with little formation of the other isomeric form of its chromophore. The alpha-band of the circular dichroism spectrum of P-495 was very small in comparison with that of rhodopsin, while that of P-466 was comparable to it. These facts suggest that P-495 has a planar conformation in the side chain of the chromophore and that P-466 has a twisted one, probably at the C8-C9 single bond. Cyclic-GMP phosphodiesterase in frog rod outer segment was activated by neither P-495 nor P-466. This result suggests that the isomerization of the retinylidene chromophore of rhodopsin is indispensable in the phototransduction process.     

Conformational similarities in the beta-ionone ring region of the rhodopsin chromophore in its ground state and after photoactivation to the metarhodopsin-I intermediate

High-resolution solid-state NMR methods have been used to analyze the conformation of the chromophore in the late photointermediate metarhodopsin-I, from observation of (13)C nuclei introduced into the beta-ionone ring (at the C16, C17, and C18 methyl groups) and into the adjoining segment of the polyene chain (at C8). Bovine rhodopsin in its native membrane was also regenerated with retinal that was (13)C-labeled close to the 11-Z bond (C20 methyl group) to provide a reporter for optimizing and quantifying the photoconversion to metarhodopsin-I. Indirect photoconversion via the primary intermediate, bathorhodopin, was adopted as the preferred method since approximately 44% conversion to the metarhodopsin-I component could be achieved, with only low levels (approximately 18%) of ground-state rhodopsin remaining. The additional photoproduct, isorhodopsin, was resolved in (13)C spectra from C8 in the chain, at levels of approximately 38%, and was shown using rotational resonance NMR to adopt the 6-s-cis conformation between the ring and the polyene chain. The C8 resonance was not shifted in the metarhodopsin-I spectral component but was strongly broadened, revealing that the local conformation had become less well defined in this segment of the chain. This line broadening slowed rotational resonance exchange with the C17 and C18 ring methyl groups but was accounted for to show that, despite the chain being more relaxed in metarhodopsin-I, its average conformation with respect to the ring was similar to that in the ground state protein. Conformational restraints are also retained for the C16 and C17 methyl groups on photoactivation, which, together with the largely preserved conformation in the chain, argues convincingly that the ring remains with strong contacts in its binding pocket prior to activation of the receptor. Previous conclusions based on photocrosslinking studies are considered in view of the current findings.     

A comparison of some photoreceptor characteristics in the pineal and retina

A rod-specific antiserum was used to immunolabel elements within the retina and pineal of the adult Djungarian hamster and Welsh Mountain sheep. In the retina immunostaining was localized to the outer segments and perikarya of photoreceptor cells, while in the pineal limited numbers of labelled pinealocytes were scattered throughout the gland. An enzyme-linked immunosorbent assay (ELISA) was then used to obtain a quantitative measure of rod opsin in total eye and pineal extracts from the Djungarian hamster. Total rod opsin (+/- SEM) in the eye was measured by absorbance spectroscopy (1.88 +/- 0.10 nmoles opsin/eye) and by using the ELISA (1.75 +/- 0.02 nmoles opsin/eye). The opsin content from a total of 56 pineals gave a mean value of 0.34 +/- 0.01 pmoles opsin/pineal. Since a functional photopigment should be coupled in a 1:1 ratio to a chromophore, we investigated whether we could identify 11-cis and/or all-trans retinaldehydes in the pineal extracts by quantitative extraction and HPLC analysis as the oximes. No evidence of 11-cis or all-trans retinaloxime could be found, the chromatograms were indistinguishable from those produced by extracts of cortical brain tissue. We conclude that the opsin present within the adult hamster pineal is not coupled to the common vertebrate retinaldehyde chromophore, and as a result, is unlikely to be part of a functional photopigment.     

Fourier transform infrared studies of active-site-methylated rhodopsin. Implications for chromophore-protein interaction, transducin activation, and the reaction pathway.

Fourier transform infrared studies of active-site-methylated rhodopsin (ASMR) show that, as compared to unmodified rhodopsin, the photoreaction is almost unchanged up to the formation of lumirhodopsin. Especially, the deviations are much smaller than those observed for the corresponding intermediates of 13-desmethyl-rhodopsin. In metarhodopsin-I, larger alterations are present with respect to the three internal carboxyl groups. Similar deviations have been observed in meta-I of 13-desmethyl-rhodopsin. This indicates that, in agreement with our previous investigations, these carboxyl groups are located in close proximity to the chromophore. Because this latter pigment is capable, when bleached, of activating transducin, our data provide support for the earlier conclusion that deprotonation of the Schiff base is a prerequisite for transducin activation. The positions of the C = C and C - C stretching modes of the retinal suggest that the redshift observed in ASMR and its photoproducts can be explained by an increased distance of the Schiff base from the counterion(s). It is further shown that the photoreaction does not stop at metarhodopsin-I, but that this intermediate directly decays to a metarhodopsin-III-like species.     

The 3, 4-didehydroretinal chromophore of goldfish porphyropsin

The isomeric configuration of the 3,4-didehydroretinal chromophore of goldfish porphyropsin was determined by high performance liquid chromatography (HPLC) and by the regeneration of this visual pigment with authentic isomers of 3,4-didehydroretinal. A nonisomerizing, quantitative method using hydroxylamine and methylene chloride was employed to extract the 3,4-didehyroretinal chromophore from the rod outer segment membrane (containing the porphyropsin). When this extracted chromophore was injected into the HPLC, only a single major peak was observed and this peak coeluted with the authentic 11-cis 3,4-didehydroretinyl oxime. This suggests that the chromophore of goldfish porphyropsin is 11-cis 3,4-didehydroretinal. When the bleached rod outer segments (containing the opsin) were incubated with different 3,4-didehydroretinal isomers (13-cis, 11-cis, 9-cis, and all-trans), only the 11-cis isomer resulted in the degeneration of porphyropsin. This also suggests that the porphyropsin chromophore exists in the 11-cis configuration.     

Chromophore structure in bacteriorhodopsin's N intermediate: implications for the proton-pumping mechanism

By elevating the pH to 9.5 in 3 M KCl, the concentration of the N intermediate in the bacteriorhodopsin photocycle has been enhanced, and time-resolved resonance Raman spectra of this intermediate have been obtained. Kinetic Raman measurements show that N appears with a half-time of 4 +/- 2 ms, which agrees satisfactorily with our measured decay time of the M412 intermediate (2 +/- 1 ms). This argues that M412 decays directly to N in the light-adapted photocycle. The configuration of the chromophore about the C13 = C14 bond was examined by regenerating the protein with [12,14-2H]retinal. The coupled C12-2H + C14-2H rock at 946 cm-1 demonstrates that the chromophore in N is 13-cis. The shift of the 1642-cm-1 Schiff base stretching mode to 1618 cm-1 in D2O indicates that the Schiff base linkage to the protein is protonated. The insensitivity of the 1168-cm-1 C14-C15 stretching mode to N-deuteriation establishes a C = N anti (trans) Schiff base configuration. The high frequency of the C14-C15 stretching mode as well as the frequency of the 966-cm-1 C14-2H-C15-2H rocking mode shows that the chromophore is 14-s-trans. Thus, N contains a 13-cis, 14-s-trans, 15-anti protonated retinal Schiff base.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrostatic interaction between retinylidene chromophore and opsin in rhodopsin studied by fluorinated rhodopsin analogues

Photochemical reactions of fluorinated rhodopsin analogues (F-rhodopsins) prepared from 10- or 12-fluorinated retinals (10- or 12-F-retinals) and cattle opsin were investigated by means of low-temperature spectrophotometry. On irradiation with blue light at liquid nitrogen temperature (-191 degrees C), the F-rhodopsins were converted to their respective batho intermediates. On warming, they decomposed to their respective fluororetinals and cattle opsin through lumi and meta intermediates. There was a difference in photochemical behavior between batho-12-F-rhodopsin and batho-10-F-rhodopsin. Upon irradiation with red light at -191 degrees C, batho-12-F-rhodopsin was converted to a mixture of 12-F-rhodopsin and 9-cis-12-F-rhodopsin like that of the natural bathorhodopsin, whereas batho-10-F-rhodopsin was not converted to 9-cis-10-F-rhodopsin but only to 10-F-rhodopsin. This fact suggests that the fluorine substituent at the C10 position (i.e., 10-fluoro) of the retinylidene chromophore may interact with the protein moiety during the process of isomerization of the chromophore or in the state of the batho intermediate. On irradiation with blue light at -191 degrees C, 9-cis-10-F-rhodopsin was converted to another bathochromic intermediate that was different in absorption spectrum from batho-10-F-rhodopsin. 9-cis-10-F-rhodopsin was practically "photoinsensitive" at liquid helium temperature (-265 degrees C), whereas 10-F-rhodopsin was converted to a photo-steady-state mixture of 10-F-rhodopsin and batho-10-F-rhodopsin. The specific interaction between the fluorine atom at the C10 position of the retinylidene chromophore and the opsin was discussed in terms of electrostatic interactions.     

Chromophore distortions in the bacteriorhodopsin photocycle: evolution of the H-C14-C15-H dihedral angle measured by solid-state NMR.

In recent years, structural information about bacteriorhodopsin has grown substantially with the publication of several crystal structures. However, precise measurements of the chromophore conformation in the various photocycle states are still lacking. This information is critical because twists about the chromophore backbone chain can influence the Schiff base nitrogen position, orientation, and proton affinity. Here, we focus on the C14-C15 bond, using solid-state nuclear magnetic resonance spectroscopy to measure the H-C14-C15-H dihedral angle. In the resting state (bR(568)), we obtain an angle of 164 +/- 4 degrees, indicating a 16 degrees distortion from a planar all-trans chromophore. The dihedral angle is found to decrease to 147 +/- 10 degrees in the early M intermediate (M(o)) and to 150 +/- 4 degrees in the late M intermediate (M(n)). These results demonstrate changes in the chromophore conformation undetected by recent X-ray diffraction studies.     

11-cis-retinal protonated Schiff base: influence of the protein environment on the geometry of the rhodopsin chromophore

Density functional theory (DFT) calculations based on the self-consistent-charge tight-binding approximation have been performed to study the influence of the protein pocket on the 3-dimensional structure of the 11-cis-retinal Schiff base (SB) chromophore. Starting with an effectively planar chromophore embedded in a protein pocket consisting of the 27 next-nearest amino acids, the relaxed chromophore geometry resulting from energy optimization and molecular dynamics (MD) simulations has yielded novel insights with respect to the following questions: (i) The conformation of the beta-ionone ring. The protein pocket tolerates both conformations, 6-s-cis and 6-s-trans, with a total energy difference of 0.7 kcal/mol in favor of the former. Of the two possible 6-s-cis conformations, the one with a negative twist angle (optimized value: -35 degrees ) is strongly favored, by 3.6 kcal/mol, relative to the one in which the dihedral is positive. (ii) Out-of-plane twist of the chromophore. The environment induces a nonplanar helical deformation of the chromophore, with the distortions concentrated in the central region of the chromophore, from C10 to C13. The dihedral angle between the planes formed by the bonds from C7 to C10 and from C13 to C15 is 42 degrees. (iii) The absolute configuration of the chromophore. The dihedral angle about the C12-C13 bond is +170 degrees from planar s-cis, which imparts a positive helicity on the chromophore, in agreement with earlier considerations based on theoretical and spectroscopic evidence.     

Assignment of fingerprint vibrations in the resonance Raman spectra of rhodopsin, isorhodopsin, and bathorhodopsin: implications for chromophore structure and environment

13C- and 2H-labeled retinal derivatives have been used to assign normal modes in the 1100-1300-cm-1 fingerprint region of the resonance Raman spectra of rhodopsin, isorhodopsin, and bathorhodopsin. On the basis of the 13C shifts, C8-C9 stretching character is assigned at 1217 cm-1 in rhodopsin, at 1206 cm-1 in isorhodopsin, and at 1214 cm-1 in bathorhodopsin. C10-C11 stretching character is localized at 1098 cm-1 in rhodopsin, at 1154 cm-1 in isorhodopsin, and at 1166 cm-1 in bathorhodopsin. C14-C15 stretching character is found at 1190 cm-1 in rhodopsin, at 1206 cm-1 in isorhodopsin, and at 1210 cm-1 in bathorhodopsin. C12-C13 stretching character is much more delocalized, but the characteristic coupling with the C14H rock allows us to assign the "C12-C13 stretch" at approximately 1240 cm-1 in rhodopsin, isorhodopsin, and bathorhodopsin. The insensitivity of the C14-C15 stretching mode to N-deuteriation in all three pigments demonstrates that each contains a trans (anti) protonated Schiff base bond. The relatively high frequency of the C10-C11 mode of bathorhodopsin demonstrates that bathorhodopsin is s-trans about the C10-C11 single bond. This provides strong evidence against the model of bathorhodopsin proposed by Liu and Asato [Liu, R., & Asato, A. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 259], which suggests a C10-C11 s-cis structure. Comparison of the fingerprint modes of rhodopsin (1098, 1190, 1217, and 1239 cm-1) with those of the 11-cis-retinal protonated Schiff base in methanol (1093, 1190, 1217, and 1237 cm-1) shows that the frequencies of the C-C stretching modes are largely unperturbed by protein binding. In particular, the invariance of the C14-C15 stretching mode at 1190 cm-1 does not support the presence of a negative protein charge near C13 in rhodopsin. In contrast, the frequencies of the C8-C9 and C14-C15 stretches of bathorhodopsin and the C10-C11 and C14-C15 stretches of isorhodopsin are significantly altered by protein binding. The implications of these observations for the mechanism of wavelength regulation in visual pigments and energy storage in bathorhodopsin are discussed.     

Effects of non-ionic surfactants N-alkyl-N,N-dimethylamine-N-oxides on the structure of a phospholipid bilayer: small-angle X-ray diffraction study

Effects of non-ionic surfactants N-alkyl-N,N-dimethylamine-N-oxides (C(n)NO, n is the number of alkyl carbons) on the structure of egg yolk phosphatidylcholine (EYPC) bilayers in the lamellar fluid phase was studied by small-angle X-ray diffraction as a function of H(2)O:EYPC and C(n)NO:EYPC molar ratios. The bilayer thickness d(L) and the lipid surface area at the bilayer-aqueous interface S(L) were calculated from the repeat period, d of the lamellar phase, based on the model that water and EYPC + CnNO molecules form separated layers and that their molecular volumes are additive. In the studied range of m=CnNO:EYPC molar ratios up to 1:1, d(L) and S(L) change linearly. The slopes Delta L = delta dL/ delta m and Delta S= delta S L / delta m are equal to -0.876 +/- 0.027 nm and 0.347 +/- 0.006 nm2 for C(6)NO, -1.025+/-0.060 nm and 0.433+/-0.025 nm(2) for C(8)NO, -0.836+/-0.046 nm and 0.405+/-0.018 nm(2) for C(10)NO, -0.604+/-0.015 nm and 0.375+/-0.007 nm(2) for C(12)NO, -0.279+/-0.031 nm and 0.318+/-0.005 nm(2) for C(14)NO, -0.0865+/-0.070 nm and 0.2963 +/-0.014 nm(2) for C(16)NO, and -0.040+/-0.022 nm and 0.297+/- 0.002 nm(2) for C(18)NO, respectively, at full bilayer hydration. The peak-peak distance in the bilayer electron density profile, which relates to the P-P distance d(PP), obtained from the first four diffraction peaks by the Fourier transform also depends linearly on m, and the slope Delta PP = delta dPP/delta m is -0.528+/-0.065 nm for C(6)NO, -0.680+/-0.018 nm for C(8)NO, -0.573+/-0.021 nm for C(10)NO, -0.369+/-0.075 nm for C(12)NO, -0.190+/-0.015 for C(14)NO, -0.088+/-0.016 nm for C(16)NO and -0.094+/-0.016 nm for C(18)NO. The effects of C(n)NO on Delta(L), Delta(S) and Delta(PP) are the results of C(n)NO insertion into EYPC bilayers and depend on the hydrophobic mismatch between C(n)NO and EYPC hydrocarbon chains and on the lateral interactions of C(n)NO and EYPC in the bilayer.