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.     

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.     

Structure of the retinal chromophore in sensory rhodopsin I from resonance Raman spectroscopy

Sensory rhodopsin I (SR-I) is a retinal-containing pigment which functions as a phototaxis receptor in Halobacterium halobium. We have obtained resonance Raman vibrational spectra of the native membrane-bound form of SR587 and used these data to determine the structure of its retinal prosthetic group. The similar frequencies and intensities of the skeletal fingerprint modes in SR587, bacteriorhodopsin (BR568), and halorhodopsin (HR578) as well as the position of the dideuterio rocking mode when SR-I is regenerated with 12,14-D2 retinal (915 cm-1) demonstrate that the retinal chromophore has an all-trans configuration. The shift of the C = N stretching mode from 1628 cm-1 in H2O to 1620 cm-1 in D2O demonstrates that the chromophore in SR587 is bound to the protein by a protonated Schiff base linkage. The small shift of the 1195 cm-1 C14-C15 stretching mode in D2O establishes that the protonated Schiff base bond has an anti configuration. The low value of the Schiff base stretching frequency together with its small 8 cm-1 shift in D2O indicates that the Schiff base proton is weakly hydrogen bonded to its protein counterion. This suggests that the red shift in the absorption maximum of SR-I (587 nm) compared with HR (578 nm) and BR (568 nm) is due to a reduction of the electrostatic interaction between the protonated Schiff base group and its protein counterion.     

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.     

Transition of rhodopsin into the active metarhodopsin II state opens a new light-induced pathway linked to Schiff base isomerization

Rhodopsin bears 11-cis-retinal covalently bound by a protonated Schiff base linkage. 11-cis/all-trans isomerization, induced by absorption of green light, leads to active metarhodopsin II, in which the Schiff base is intact but deprotonated. The subsequent metabolic retinoid cycle starts with Schiff base hydrolysis and release of photolyzed all-trans-retinal from the active site and ends with the uptake of fresh 11-cis-retinal. To probe chromophore-protein interaction in the active state, we have studied the effects of blue light absorption on metarhodopsin II using infrared and time-resolved UV-visible spectroscopy. A light-induced shortcut of the retinoid cycle, as it occurs in other retinal proteins, is not observed. The predominantly formed illumination product contains all-trans-retinal, although the spectra reflect Schiff base reprotonation and protein deactivation. By its kinetics of formation and decay, its low temperature photointermediates, and its interaction with transducin, this illumination product is identified as metarhodopsin III. This species is known to bind all-trans-retinal via a reprotonated Schiff base and forms normally in parallel to retinal release. We find that its generation by light absorption is only achieved when starting from active metarhodopsin II and is not found with any of its precursors, including metarhodopsin I. Based on the finding of others that metarhodopsin III binds retinal in all-trans-C(15)-syn configuration, we can now conclude that light-induced formation of metarhodopsin III operates by Schiff base isomerization ("second switch"). Our reaction model assumes steric hindrance of the retinal polyene chain in the active conformation, thus preventing central double bond isomerization.     

Strongly hydrogen-bonded water molecule present near the retinal chromophore of Leptosphaeria rhodopsin, the bacteriorhodopsin-like proton pump from a eukaryote

Leptosphaeria rhodopsin (LR) is an archaeal-type rhodopsin found in fungi, and is the first light-driven proton-pumping retinal protein from eukaryotes. LR pumps protons in a manner similar to that of bacteriorhodopsin (BR), a light-driven proton pump of haloarchaea. The amino acid sequence of LR is more homologous to that of Neurospora rhodopsin (NR) than BR, whereas NR has no proton-pumping activity. These facts raise the question of how the proton-pumping function is achieved. In this paper, we studied structural changes of LR following the retinal photoisomerization by means of low-temperature Fourier transform infrared (FTIR) spectroscopy, and compared the obtained spectra with those for BR and NR. While the light-induced photoisomerization from the all-trans to 13-cis form was commonly observed among LR, BR, and NR, we found that the structural changes of LR are closer to those of BR than to those of NR in terms of detailed vibrational bands of retinal and protein. The most prominent difference was seen for the water O-D stretching vibrations (measured in D2O). LR exhibits an O-D stretch of water at 2257 cm(-1), indicating the presence of a strongly hydrogen-bonded water molecule. Such strongly hydrogen-bonded water molecules (O-D stretch at <2400 cm(-1)) were observed for BR, but not for NR. Comprehensive studies of BR mutants and archaeal rhodopsins have revealed that strongly hydrogen-bonded water molecules are found only in the proteins exhibiting proton-pumping activity, suggesting that strongly hydrogen-bonded water molecules and transient weakening of their binding are essential for the proton-pumping function of rhodopsins. This observation for LR provided additional experimental evidence of the correlation between strongly hydrogen-bonded water molecules and proton-pumping activity of archaeal rhodopsins.     

Molecular dynamics simulations of retinal in rhodopsin: from the dark-adapted state towards lumirhodopsin

The formation of photointermediates and conformational changes observed in the retinal chromophore of bilayer-embedded rhodopsin during the early steps of the protein activation have been studied by molecular dynamics (MD) simulation. In particular, the lysine-bound retinal has been examined, focusing on its conformation in the dark-adapted state (10 ns) and on the early steps after the isomerization of the 11-cis bond to trans (up to 10 ns). The parametrization for the chromophore is based on a recent quantum study [Sugihara, M., Buss, V., Entel, P., Elstner, M., and Frauenheim, T. (2002) Biochemistry 41, 15259-15266] and shows good conformational agreement with recent experimental results. The isomerization, induced by switching the function governing the dihedral angle for the C11=C12 bond, was repeated with several different starting conformations. From the repeated simulations, it is shown that the retinal model exhibits a conserved activation pattern. The conformational changes are sequential and propagate outward from the C11=C12 bond, starting with isomerization of the C11=C12 bond, then a rotation of methyl group C20, and followed by increased fluctuations at the beta-ionone ring. The dynamics of these changes suggest that they are linked with photointermediates observed by spectroscopy. The exact moment when these events occur after the isomerization is modulated by the starting conformation, suggesting that retinal isomerizes through multiple pathways that are slightly different. The amplitudes of the structural fluctuations observed for the protein in the dark-adapted state and after isomerization of the retinal are similar, suggesting a subtle mechanism for the transmission of information from the chromophore to the protein.     

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.     

The molecular origin of the inhibition of transducin activation in rhodopsin lacking the 9-methyl group of the retinal chromophore: a UV-Vis and FTIR spectroscopic study

The formation of the active rhodopsin state metarhodopsin II (MII) is believed to be partially governed by specific steric constraints imposed onto the protein by the 9-methyl group of the retinal chromophore. We studied the properties of the synthetic pigment 9-demethyl rhodopsin (9dm-Rho), consisting of the rhodopsin apoprotein regenerated with synthetic retinal lacking the 9-methyl group, by UV-vis and Fourier transform infrared difference spectroscopy. Low activation rates of the visual G-protein transducin by the modified pigment reported in previous studies are shown to not be caused by the reduced activity of its MII state, but to be due to a dramatic equilibrium shift from MII to its immediate precursor, MI. The MII state of 9dm-Rho displays only a partial deprotonation of the retinal Schiff base, leading to the formation of two MII subspecies absorbing at 380 and 470 nm, both of which seem to be involved in transducin activation. The rate of MII formation is slowed by 2 orders of magnitude compared to rhodopsin. The dark state and the MI state of 9dm-Rho are distinctly different from their respective states in the native pigment, pointing to a more relaxed fit of the retinal chromophore in its binding pocket. The shifted equilibrium between MI and MII is therefore discussed in terms of an increased entropy of the 9dm-Rho MI state due to changed steric interactions.     

Elements in the C terminus of apolipoprotein [a] responsible for the binding to the tenth type III module of human fibronectin

In previous studies, we showed that the C-terminal domain, F2, but not the N-terminal domain, F1, is responsible for the binding of apolipoprotein [a] (apo[a]) to human fibronectin (Fn). To pursue those observations, we prepared, by both elastase digestion and recombinant technology, subsets of F2 of a different length containing either kringle (K) V or the protease domain (PD). We also studied rhesus monkey apo[a], which is known to contain PD but not KV. In the case of Fn, we used both an intact product and its tenth type III module (10FN-III) expressed in Escherichia coli. The binding studies carried out on microtiter plates showed that the affinity of F2 for immobilized 10FN-III was approximately 6-fold higher than that for Fn (dissociation constants = 1.75 +/- 0.31 nM and 10.25 +/- 1.62 nM, respectively). The binding was also exhibited by rhesus apo[a] and by an F2 subset containing the PD linked to an upstream microdomain comprising KIV-8 to KIV-10 and KV, inactive by itself. Competition experiments on microtiter plates showed that both Fn and 10FN-III, when in solution, are incompetent to bind F2. Together, our results indicate that F2 binds to immobilized 10FN-III more efficiently than whole Fn and that the binding can be sustained by truncated forms of F2 that contain the catalytically inactive PD linked to an upstream four K microdomain.     

Evidence for cis-trans isomerization of a double bond in the fatty acids of the psychrophilic bacterium Vibrio sp. strain ABE-1.

Vibrio sp. strain ABE-1 was grown in a medium that contained as its stable isotope tracer either [2,2-2H2]cis-9-hexadecenoic or [2,2-2H2]trans-9-hexadecenoic acid. Gas chromatographic-mass spectrometric analysis of the cis-9-hexadecenoic and trans-9-hexadecenoic acid fractions from the cells revealed the formation of an intracellularly isomerized 2,2-2H2-fatty acid which differed from the tracer only in the geometrical configuration of the double bond. This observation shows that cis-trans isomerization without a shift in double-bond position between these two geometric hexadecenoic acid isomers can occur in the cells.     

Irreversible thermal denaturation of glucose oxidase from Aspergillus niger is the transition to the denatured state with residual structure

Glucose oxidase (GOX; beta-d-glucose:oxygen oxidoreductase) from Aspergillus niger is a dimeric flavoprotein with a molecular mass of 80 kDa/monomer. Thermal denaturation of glucose oxidase has been studied by absorbance, circular dichroism spectroscopy, viscosimetry, and differential scanning calorimetry. Thermal transition of this homodimeric enzyme is irreversible and, surprisingly, independent of GOX concentration (0.2-5.1 mg/ml). It has an apparent transition temperature of 55.8 +/- 1.2 degrees C and an activation energy of approximately 280 kJ/mol, calculated from the Lumry-Eyring model. The thermally denatured state of GOX after recooling has the following characteristics. (i) It retains approximately 70% of the native secondary structure ellipticity; (ii) it has a relatively low intrinsic viscosity, 7.5 ml/g; (iii) it binds ANS; (iv) it has a low Stern-Volmer constant of tryptophan quenching; and (v) it forms defined oligomeric (dimers, trimers, tetramers) structures. It is significantly different from chemically denatured (6.67 m GdmHCl) GOX. Both the thermal and the chemical denaturation of GOX cause dissociation of the flavin cofactor; however, only the chemical denaturation is accompanied by dissociation of the homodimeric GOX into monomers. The transition temperature is independent of the protein concentration, and the properties of the thermally denatured protein indicate that thermally denatured GOX is a compact structure, a form of molten globule-like apoenzyme. GOX is thus an exceptional example of a relatively unstable mesophilic dimeric enzyme with residual structure in its thermally denatured state.     

Identification of activities that catalyze the cis-trans isomerization of the double bond of a mono-unsaturated fatty acid in Pseudomonas sp. strain E-3

A cell-free extract of Pseudomonas sp. strain E-3 catalyzed the conversion of 9-cis-hexadecenoic acid [16:1(9c)] to 9-trans-hexadecenoic acid [16:1(9t)] in the free acid form and when 16:1(9c) was esterified to phosphatidylethanolamine (PE). The cytosolic fraction catalyzed the isomerizations of free 16:1(9c) by itself and of 16:1(9c) esterified to PE in the presence of the membrane fraction. Tracer experiments using [2,2-²H₂]16:1(9c) demonstrated that the isomerization of free 16:1(9c) occurred independently of the isomerization of 16:1(9c) esterified to PE, indicating that this bacterium has two types of activities that catalyze the cis-trans isomerization of the double bond of a mono-unsaturated fatty acid. Received: 29 December 1995 / Accepted: 10 April 1996     

Capturing the photo‐signaling state of a photoreceptor in a steady‐state fashion by binding a transition metal complex

Binding a small molecule to proteins causes conformational changes, but often to a limited extent. Here, we demonstrate that the interaction of a CO‐releasing molecule (CORM3) with a photoreceptor photoactive yellow protein (PYP) drives large structural changes in the latter. The interaction of CORM3 and a mutant of PYP, Met100Ala, not only trigger the isomerization of its chromophore, p‐coumaric acid, from its anionic trans configuration to a protonated cis configuration, but also increases the content of β‐sheet at the cost of α‐helix and random coil in the secondary structure of the protein. The CORM3 derived Met100Ala is found to highly resemble the signaling state, which is one of the key photo‐intermediates of this photoactive protein, in both protein local conformation and chromophore configuration. The organometallic reagents hold promise as protein engineering tools. This work highlights a novel approach to structurally accessing short lived intermediates of proteins in a steady‐state fashion.     

Fertilization initiates the transition from anaphase I to metaphase II during female meiosis in C. elegans

Oocytes from most animals arrest twice during the meiotic cell cycle. The universally conserved prophase I arrest is released by a maturation hormone that allows progression to a second arrest point, typically metaphase I or II. This second arrest allows for short-term storage of fertilization-competent eggs and is released by signaling that occurs during fertilization. Nematodes are unique in that the maturation hormone is secreted by sperm rather than by the mother's somatic tissues. We have investigated the nature of the second arrest in matured but unfertilized Caenorhabditis elegans embryos using time-lapse imaging of GFP-tubulin or GFP-histone. Unfertilized embryos completed anaphase I but did not form polar bodies or assemble meiosis II spindles. Nevertheless, unfertilized embryos assembled female pronuclei at the same time as fertilized embryos. Analysis of embryos fertilized by sperm lacking the SPE-11 protein indicated that fertilization promotes meiotic cytokinesis through the SPE-11 protein but assembly of the meiosis II spindle is initiated through an SPE-11-independent pathway.     

A theoretical investigation of the intercalative binding of 7-H pyrido[4.3C]carbazole chromophore into a d(CpG)2 minihelix

Theoretical computations are performed of the intercalative binding to a model d(CpG)2 minihelix of 7-H pyrido[4.3C]carbazole, the precursor of the antitumor bisintercalating drug ditercalinium. The conformations of the intercalation site are generated by the AGNAS procedure (algorithm to generate nucleic acid structures) of Miller and co-workers. The ligand-nucleotide interactions and the nucleotide conformational energies are computed with the SIBFA procedures (sum of interactions between fragments ab initio computed), which use formulas of empirical origin that reproduce ab initio SCF (self-consistent field) computations. Among the candidate intercalation sites most favored energetically, one has a pattern of conformational angles related to the one determined crystallographically by Sobell et al. in a series of x-ray structural studies of small intercalator-dinucleotide monophosphate complexes. Optimal values of the unwinding angle, found in the range of -12 degrees to -14 degrees, are consistent with available experimental data on DNA.     

Electrostatic coupling between retinal isomerization and the ionization state of Glu-204: a general mechanism for proton release in bacteriorhodopsin.

The pKa values of ionizable groups that lie between the active site region of bacteriorhodopsin (bR) and the extracellular surface of the protein are reported. Glu-204 is found to have an elevated pKa in the resting state of bR, suggesting that it corresponds to the proton-releasing group in bR. Its elevated pKa is predicted to be due in part to strong repulsive interactions with Glu-9. Following trans-cis isomerization of the retinal chromophore and the transfer of a proton to Asp-85, polar groups on the protein are able to interact more strongly with the ionized state of Glu-204, leading to a substantial reduction of its pKa. This suggests a general mechanism for proton release in which isomerization and subsequent charge separation initially produce a new electrostatic balance in the active site of bR. Here it is proposed that those events in turn drives a conformational change in the protein in which the ionized state of Glu-204 can be stabilized through interactions with groups that were previously inaccessible. Whether these groups should be identified with polar moieties in the protein, bound waters, or Arg-82 is an important mechanistic question whose elucidation will require further study.     

Generation of C3- and C2-deuterated l-lactic acid by human erythrocytes exposed to d-[1-13C]glucose,d-[2-13c]glucose and d-[6-13C]glucose in the presence of D2O

• 1.1. The generation of C₂- and C₃-deuterated l-lactate was monitored by ¹³C NMR in human erythrocytes exposed to d-[1-¹³glucose, d-[2-¹³C]glucose or d-te-¹³C]glucose and incubated in a medium prepared in D₂O.
• 2.2. The results suggested that the deuteration of the C₁ of d-fructose 6-phosphate in the phosphoglucoisomerase reaction, the deuteration of the C₁ of d-glyceraldehyde-3-phosphate in the sequence of reactions catalyzed by triose phosphate isomerase and aldolase and the deuteration of the C₃ of pyruvate in the reaction catalyzed by pyruvate kinase were all lower than expected from equilibration with D₂O.
• 3.3. Moreover, about 40% of the molecules of pyruvate generated by glycolysis apparently underwent deuteration on their C₃ during interconversion of the 2-keto acid and l-alanine in the reaction catalyzed by glutamate-pyruvate transaminase.
• 4.4. The occurrence of the latter process was also documented in cells exposed to exogenous [3-¹³C]pyruvate.
• 5.5. This methodological approach is proposed to provide a new tool to assess in intact cells the extent of back-and-forth interconversion of selected metabolic intermediates.