Protonated Schiff Base Research Articles

Colour tuning mechanisms of visual pigments.

Spectral tuning by visual pigments involves modulation of physical properties of the 11-cis-retinylidene protonated Schiff base (PSB) chromophore by amino acid side chains in and around the chromophore-binding pocket. Specific molecular contacts between the chromophore and the amino acid side chains of the opsin chromophore-binding pocket have been determined recently using an interdisciplinary approach consisting of site-directed mutagenesis, optical and vibrational spectroscopy, and molecular graphics modelling. These studies provide insight into the mechanism of spectral tuning among visual pigments. In blue pigments a majority of the opsin shift is caused by polar amino acid side chains arrayed about the PSB to increase the energy gap between the ground (S0) and excited states (S1). In addition, a specific tyrosine near the chromophore ring causes a decrease in solvent polarizability. Other amino acid residues alter the binding pocket structure to strengthen electrostatic interaction between the PSB and its counterion and/or solvent dipoles. In the green and red pigments, the work of Kochendoerfer et al (1997; Biochemistry 26:6577-6587) demonstrates that local structural perturbations at the PSB or elsewhere are not responsible for spectral tuning. Instead, the green-to-red opsin shift is best explained by dipolar side chains near the chromophore ring that lower the transition energy that occurs upon electronic excitation by affecting the change in electric dipole moment. In summary, the absorption maximum of a visual pigment is primarily regulated by the interaction of the chromophore charge distribution with dipolar residues in its opsin chromophore-binding pocket. The work presented in this paper is reported in greater detail in Lin et al.

Photons, femtoseconds and dipolar interactions: a molecular picture of the primary events in vision.

The first 200 femtoseconds in the life of a photoexcited rhodopsin molecule are extremely important for the development of visual sensation. Immediately upon excitation, a dramatic change in the charge distribution of the cationic 11-cis-retinal protonated Schiff base chromophore occurs that is quantitated by the change in electronic dipole moment of approximately 15 Debye. The opsin protein tunes the absorption maximum of the pigment to the blue or to the red enabling colour vision by placing dipolar rather than charged residues in the chromophore binding site to differentially stabilize either the ground or the excited state charge distribution. Resonance Raman intensity analysis reveals that the 11-cis-retinal chromophore then distorts violently about the C11 = C12 double bond, reaching torsional angles of approximately 70 degrees in only 30 fs. This rapid torsional distortion is driven by the non-bonded interaction between the 13-methyl group and the 10-hydrogen that is unique to the 11-cis configuration of the chromophore. The excited state depopulates in approximately 50 fs through a rapid and vibrationally coherent transition to the ground electronic state manifold with relaxation to the formally trans photoproduct complete in only 200 fs. This unusually fast and efficient isomerization process establishes a new paradigm for condensed phase photochemical reactions.

Structure Elucidation of Dyes That Are Formed in the Colorimetric Detection of the Improvised Explosive Urea Nitrate

Urea nitrate (uronium nitrate, UN) is a powerful, improvised explosive that can be easily made from urea and nitric acid. It is considered the most frequently used, illegal explosive in the Israeli arena, which is responsible for the loss of more than a hundred lives in terrorist incidents. Urea nitrate is a colorless, crystalline substance that looks very much like sugar. A sensitive color test for UN was developed recently. It is based on the formation of a red dye in the reaction between p-dimethylaminocinnamaldehyde and UN under neutral conditions. A similar reaction with p-dimethylaminobezaldehyde produces a yellow dye. The two dyes have been synthesized, and their structures determined by X-ray crystallography. Both dyes are protonated Schiff bases, prevailing in the crystal in a quinoid form. They are identical to the compounds, which are obtained in the colorimetric detection of urea with the same reagents, under strong acidic conditions, whose structures have been postulated in the literature, but never fully proved experimentally.

Stairway to the Conical Intersection: A Computational Study of the Retinal Isomerization

The potential-energy surface of the first excited state of the 11-cis-retinal protonated Schiff base (PSB11) chromophore has been studied at the density functional theory (DFT) level using the time-dependent perturbation theory approach (TDDFT) in combination with Becke's three-parameter hybrid functional (B3LYP). The potential-energy curves for torsion motions around single and double bonds of the first excited state have also been studied at the coupled-cluster approximate singles and doubles (CC2) level. The corresponding potential-energy curves for the ground state have been calculated at the B3LYP DFT and second-order Møller-Plesset (MP2) levels. The TDDFT study suggests that the electronic excitation initiates a turn of the beta-ionone ring around the C6-C7 bond. The torsion is propagating along the retinyl chain toward the cis to trans isomerization center at the C11=C12 double bond. The torsion twist of the C10-C11 single bond leads to a significant reduction in the deexcitation energy indicating that a conical intersection is being reached by an almost barrierless rotation around the C10-C11 single bond. The energy released when passing the conical intersection can assist the subsequent cis to trans isomerization of the C11=C12 double bond. The CC2 calculations also show that the torsion barrier for the twist of the retinyl C10-C11 single bond adjacent to the isomerization center almost vanishes for the excited state. Because of the reduced torsion barriers of the single bonds, the retinyl chain can easily deform in the excited state. Thus, the CC2 and TDDFT calculations suggest similar reaction pathways on the potential-energy surface of the excited state leading toward the conical intersection and resulting in a cis to trans isomerization of the retinal chromophore. According to the CC2 calculations the cis to trans isomerization mechanism does not involve any significant torsion motion of the beta-ionone ring.

Di-μ-chlorido-bis(chlorido{2-[(2-hydroxyphenyl)iminiomethyl]-6-methoxyphenolato-κ2O,O′}cadium(II)) methanol disolvate

The title centrosymmetric complex, [Cd2Cl4(C14H13NO3)2]·2CH3OH, contains two protonated Schiff base ligands which were derived from the condensation of o-vanillin and 2-hydroxy­aniline. The CdII ion is five-coordinated by three Cl atoms and two O atoms in a distorted square-pyramidal geometry. A basal edge containing two bridging chloride ligands is shared by both symmetry-related CdII ions. The four atoms of the Cd2Cl2 core are coplanar, giving a rhomboidal geometry with two short and two long Cd—Cl distances and acute Cl—Cd—Cl and obtuse Cd—Cl—Cd angles. The crystal structure is stabilized by inter­molecular hydrogen bonds and weak π–π stacking inter­actions with a centroid-to-centroid distance of 3.710 (4) Å.

Vibrational coherences of the protonated Schiff base of all-trans retinal in solution

Modulations in the time gated ultrafast fluorescence of the protonated Schiff base of retinal in various solvents are reported, which reflect the creation and evolution of vibrational wave packets due to the torsional mode of retinal of frequency ∼120 cm −1. The oscillations are damped in ∼400 fs. Their frequency is significantly decreased compared to the case of the protein (170 cm −1), and is insensitive to solvent properties such as the dielectric constant and the viscosity. This, along with previous results on mutants of bacteriorhodopsin, leads us to conclude that in the protein, the isomerization dynamics of retinal is governed by steric effects and by the inhomogeneous distribution of the electrostatic field due to amino-acid residues within the protein pocket. We also discuss the origin of the modulations and conclude that they are impulsively excited via the high frequency modes of retinal.

Halide Binding by the D212N Mutant of Bacteriorhodopsin Affects Hydrogen Bonding of Water in the Active Site

Bacteriorhodopsin (BR), a membrane protein found in Halobacterium salinarum, functions as a light-driven proton pump. The Schiff base region has a quadrupolar structure with positive charges located at the protonated Schiff base and Arg82, and the counterbalancing negative charges located at Asp85 and Asp212. The quadropole inside the protein is stabilized by three water molecules, forming a roughly planar pentagonal cluster composed of these waters and two oxygens of Asp85 and Asp212 (one from each carboxylate side chain). It is known that BR lacks proton-pumping activity if Asp85 or Asp212 is neutralized by mutation, but binding of Cl- has different functional effects in mutants at these positions. Binding of Cl- to D85T converts into a chloride ion pump (Sasaki, J., Brown, L. S., Chon, Y.-S., Kandori, H., Maeda, A., Needleman, R., and Lanyi, J. K. (1995) Science 269, 73-75). On the other hand, photovoltage measurements suggested that binding of Cl- to D212N restores the proton-pumping activity at low pH (Moltke, S., Krebs, M. P., Mollaaghababa, R., Khorana, H. G., and Heyn, M. P. (1995) Biophys. J. 69, 2074-2083). In this paper, we studied halide-bound D212N mutant BR in detail. Light-induced pH changes in a suspension of proteoliposomes containing D212N(Cl-) at pH 5 clearly showed that Cl- restores the proton-pumping activity. Spectral blue-shift induced by halide binding to D212N indicates that halides affect the counterion of the protonated Schiff base, whereas much smaller halide dependence of the lambdamax than in D85T suggests that the binding site is distant from the chromophore. In fact, the K minus BR difference Fourier-transform infrared (FTIR) spectra of D212N at 77 K exhibit little halide dependence for vibrational bands of retinal and protein. The only halide-dependent bands were the C=N stretch of Arg82 and some water O-D stretches, suggesting that these groups constitute a halide-binding pocket. A strongly hydrogen-bonded water molecule is observed for halide-bound D212N, but not for halide-free D212N, which is consistent with our hypothesis that such a water molecule is a prerequisite for proton-pumping activity of rhodopsins. We concluded that halide binding near Arg82 in D212N restores the water-containing hydrogen-bonding network in the Schiff base region. In particular, the ion pair formed by the Schiff base and Asp85 through a strongly hydrogen-bonded water is essential for the proton-pumping activity of this mutant and may be controlled by the halide binding to the distant site.

Photoisomerization Efficiency in UV-Absorbing Visual Pigments: Protein-Directed Isomerization of an Unprotonated Retinal Schiff Base

A visual pigment consists of an opsin protein and a chromophore, 11-cis-retinal, which binds to a specific lysine residue of opsin via a Schiff base linkage. The Schiff base chromophore is protonated in pigments that absorb visible light, whereas it is unprotonated in ultraviolet-absorbing visual pigments (UV pigments). To investigate whether an unprotonated Schiff base can undergo photoisomerization as efficiently as a protonated Schiff base in the opsin environment, we measured the quantum yields of the bovine rhodopsin E113Q mutant, in which the Schiff base is unprotonated at alkaline pH, and the mouse UV pigment (mouse UV). Photosensitivities of UV pigments were measured by irradiation of the pigments followed by chromophore extraction and HPLC analysis. Extinction coefficients were estimated by comparing the maximum absorbances of the original pigments and their acid-denatured states. The quantum yield of the bovine rhodopsin E113Q mutant at pH 8.2, where the Schiff base is unprotonated, was significantly lower than that of wild-type rhodopsin, whereas the mutant gave a quantum yield almost identical to that of the wild type at pH 5.5, where the Schiff base is protonated. These results suggest that Schiff base protonation plays a role in increasing quantum yield. The quantum yield of mouse UV, which has an unprotonated Schiff base chromophore, was significantly higher than that of the unprotonated form of the rhodopsin E113Q mutant, although it was still lower than the visible-absorbing pigments. These results suggest that the mouse UV pigment has a specific mechanism for the efficient photoisomerization of its unprotonated Schiff base chromophore.

Structure of the Conical Intersections Driving the cis-trans Photoisomerization of Conjugated Molecules¶

High-level ab initio calculations show that the singlet photochemical cis–trans isomerization of organic molecules under isolated conditions can occur according to two distinct mechanisms. These mechanisms are characterized by the different structures of the conical intersection funnels controlling photoproduct formation. In nonpolar (e.g. hydrocarbon) polyenes the lowest-lying funnel corresponds to a (CH)3 kink with both double and adjacent single bonds twisted, which may initiate hula-twist (HT) isomerization. On the other hand, in polar conjugated systems such as protonated Schiff bases (PSB) the funnel shows a structure with just one twisted double bond. The ground-state relaxation paths departing from the funnels indicate that the HT motion may take place in nonpolar conjugated systems but also that the single-bond twist may be turned back, whereas in free conjugated polar molecules such as PSB a one-bond flip mechanism dominates from the beginning. The available experimental evidence either supports these predictions or is at least consistent with them.

Histidine 282 in 5-Aminolevulinate Synthase Affects Substrate Binding and Catalysis

5-Aminolevulinate synthase (ALAS), the first enzyme of the heme biosynthetic pathway in mammalian cells, is a member of the alpha-oxoamine synthase family of pyridoxal 5'-phosphate (PLP)-dependent enzymes. In all structures of the enzymes of the -oxoamine synthase family, a conserved histidine hydrogen bonds with the phenolic oxygen of the PLP cofactor and may be significant for substrate binding, PLP positioning, and maintenance of the pKa of the imine nitrogen. In ALAS, replacing the equivalent histidine, H282, with alanine reduces the catalytic efficiency for glycine 450-fold and decreases the slow phase rate for glycine binding by 85%. The distribution of the absorbing 420 and 330 nm species was altered with an A420/A330 ratio increased from 0.45 to 1.05. This shift in species distribution was mirrored in the cofactor fluorescence and 300-500 nm circular dichroic spectra and likely reflects variation in the tautomer distribution of the holoenzyme. The 300-500 nm circular dichroism spectra of ALAS and H282A diverged in the presence of either glycine or aminolevulinate, indicating that the reorientation of the PLP cofactor upon external aldimine formation is impeded in H282A. Alterations were also observed in the K(Gly)d value and spectroscopic and kinetic properties, while the K(PLP)d increased 9-fold. Altogether, the results imply that H282 coordinates the movement of the pyridine ring with the reorganization of the active site hydrogen bond network and acts as a hydrogen bond donor to the phenolic oxygen to maintain the protonated Schiff base and enhance the electron sink function of the PLP cofactor.

Critical Role of Electrostatic Interactions of Amino Acids at the Cytoplasmic Region of Helices 3 and 6 in Rhodopsin Conformational Properties and Activation

The cytoplasmic sides of transmembrane helices 3 and 6 of G-protein-coupled receptors are connected by a network of ionic interactions that play an important role in maintaining its inactive conformation. To investigate the role of such a network in rhodopsin structure and function, we have constructed single mutants at position 134 in helix 3 and at positions 247 and 251 in helix 6, as well as combinations of these to obtain double mutants involving the two helices. These mutants have been expressed in COS-1 cells, immunopurified using the rho-1D4 antibody, and studied by UV-visible spectrophotometry. Most of the single mutations did not affect chromophore formation, but double mutants, especially those involving the T251K mutant, resulted in low yield of protein and impaired 11-cis-retinal binding. Single mutants E134Q, E247Q, and E247A showed the ability to activate transducin in the dark, and E134Q and E247A enhanced activation upon illumination, with regard to wild-type rhodopsin. Mutations E247A and T251A (in E134Q/E247A and E134Q/T251A double mutants) resulted in enhanced activation compared with the single E134Q mutant in the dark. A role for Thr(251) in this network is proposed for the first time in rhodopsin. As a result of these mutations, alterations in the hydrogen bond interactions between the amino acid side chains at the cytoplasmic region of transmembrane helices 3 and 6 have been observed using molecular dynamics simulations. Our combined experimental and modeling results provide new insights into the details of the structural determinants of the conformational change ensuing photoactivation of rhodopsin.

Coupling of Protonation Switches During Rhodopsin Activation†

Recent studies of the activation mechanism of rhodopsin involving Fourier-transform infrared spectroscopy and a combination of chromophore modifications and site-directed mutagenesis reveal an allosteric coupling between two protonation switches. In particular, the ring and the 9-methyl group of the all-trans retinal chromophore serve to couple two proton-dependent activation steps: proton uptake by a cytoplasmic network between transmembrane (TM) helices 3 and 6 around the conserved ERY (Glu-Arg-Tyr) motif and disruption of a salt bridge between the retinal protonated Schiff base (PSB) and a protein counterion in the TM core of the receptor. Retinal analogs lacking the ring or 9-methyl group are only partial agonists--the conformational equilibrium between inactive Meta I and active Meta II photoproduct states is shifted to Meta I. An artificial pigment was engineered, in which the ring of retinal was removed and the PSB salt bridge was weakened by fluorination of C14 of the retinal polyene. These modifications abolished allosteric coupling of the proton switches and resulted in a stabilized Meta I state with a deprotonated Schiff base (Meta I(SB)). This state had a partial Meta II-like conformation due to disruption of the PSB salt bridge, but still lacked the cytoplasmic proton uptake reaction characteristic of the final transition to Meta II. As activation of native rhodopsin is known to involve deprotonation of the retinal Schiff base prior to formation of Meta II, this Meta I(SB) state may serve as a model for the structural characterization of a key transient species in the activation pathway of a prototypical G protein-coupled receptor.

Protein Design: Reengineering Cellular Retinoic Acid Binding Protein II into a Rhodopsin Protein Mimic

Rational redesign of the binding pocket of Cellular Retinoic Acid Binding Protein II (CRABPII) has provided a mutant that can bind retinal as a protonated Schiff base, mimicking the binding observed in rhodopsin. The reengineering was accomplished through a series of choreographed manipulations to ultimately orient the reactive species (the epsilon-amino group of Lys132 and the carbonyl of retinal) in the proper geometry for imine formation. The guiding principle was to achieve the appropriate Bürgi-Dunitz trajectory for the reaction to ensue. Through crystallographic analysis of protein mutants incapable of forming the requisite Schiff base, a highly ordered water molecule was identified as a key culprit in orienting retinal in a nonconstructive manner. Removal of the ordered water, along with placing reinforcing mutations to favor the desired orientation of retinal, led to a triple mutant CRABPII protein capable of nanomolar binding of retinal as a protonated Schiff base. The high-resolution crystal structure of all-trans-retinal bound to the CRABPII triple mutant (1.2 A resolution) unequivocally illustrates the imine formed between retinal and the protein.

FTIR Study of the Retinal Schiff Base and Internal Water Molecules of Proteorhodopsin

Proteorhodopsin (PR), an archaeal-type rhodopsin found in marine bacteria, is a light-driven proton pump similar to bacteriorhodopsin (BR). It is known that Asp97, a counterion of the protonated Schiff base, possesses a higher pKa ( approximately 7) compared to that of homologous Asp85 in BR (<3). This suggests that PR has a hydrogen-bonding network different from that of BR. We previously reported that a strongly hydrogen-bonded water molecule is observed only in the alkaline form of PR, where Asp97 is deprotonated (Furutani, Y., Ikeda, D., Shibata, M., and Kandori, H. (2006) Chem. Phys. 324, 705-708). This is probably correlated with the pH-dependent proton pumping activity of PR. In this work, we studied the water-containing hydrogen-bonding network in the Schiff base region of PR by means of Fourier-transform infrared (FTIR) spectroscopy at 77 K. [zeta-15N]Lys-labeling and 18O water were used for assigning the Schiff base N-D and water O-D stretching vibrations in D2O, respectively. The frequency upshift of the N-D stretch in the primary K intermediate is much smaller for PR than for BR, indicating that the Schiff base forms a hydrogen bond after retinal photoisomerization. We then measured FTIR spectra of the mutants of Asp97 (D97N and D97E) and Asp227 (D227N and D227E) to identify the amino acid interacting with the Schiff base in the K state. The PRK minus PR spectra of D97N and D97E were similar to those of the acidic and alkaline forms, respectively, of the wild type implying that the structural changes upon retinal photoisomerization are not influenced by the mutation at Asp97. In contrast, clear spectral differences were observed in D227N and D227E, including vibrational bands of the Schiff base and water molecules. It is concluded that Asp227 plays a crucial role during the photoisomerization process, though Asp97 acts as the primary counterion in the unphotolyzed state of PR.

Pressure-induced Isomerization of Retinal on Bacteriorhodopsin as Disclosed by Fast Magic Angle Spinning NMR†

Bacteriorhodopsin (bR) is a retinal protein in purple membrane of Halobacterium salinarum, which functions as a light-driven proton pump. We have detected pressure-induced isomerization of retinal in bR by analyzing 15N cross polarization-magic angle spinning (CP-MAS) NMR spectra of [zeta-15N]Lys-labeled bR. In the 15N-NMR spectra, both all-trans and 13-cis retinal configurations have been observed in the Lys N(zeta) in protonated Schiff base at 148.0 and 155.0 ppm, respectively, at the MAS frequency of 4 kHz in the dark. When the MAS frequency was increased up to 12 kHz corresponding to the sample pressure of 63 bar, the 15N-NMR signals of [zeta-15N]Lys in Schiff base of retinal were broadened. On the other hand, other [zeta-15N]Lys did not show broadening. Subsequently, the increased signal intensity of [zeta-15N]Lys in Schiff base of 13-cis retinal at 155.0 ppm was observed when the MAS frequency was decreased from 12 to 4 kHz. These results showed that the equilibrium constant of [all-trans-bR]/[13-cis-bR] in retinal decreased by the pressure of 63 bar. It was also revealed that the structural changes induced by the pressure occurred in the vicinity of retinal. Therefore, microscopically, hydrogen-bond network around retinal would be disrupted or distorted by a constantly applied pressure. It is, therefore, clearly demonstrated that increased pressure induced by fast MAS frequencies generated isomerization of retinal from all-trans to 13-cis state in the membrane protein bR.

About the intrinsic photochemical properties of the 11-cis retinal chromophore: computational clues for a trap state and a lever effect in Rhodopsin catalysis

CASPT2//CASSCF/6-31G* computations are used on the singlet S 1 and S 2 states to map the photoisomerization process of the 11-cis retinal protonated Schiff base in vacuo and to characterize its optical properties. It is shown that the spectroscopic observations recorded in Rhodopsin are reproduced quite well, calling for a substantially neutral effect of the protein. Furthermore, a rationale is proposed for the unreactive population recently observed in Rhodopsin, which is here addressed to the accessible S 2 state, behaving as a trap. The experimental transient absorption and (absorption-wavelength dependent) emission are discussed and interpreted under the light of this novel model. Finally, a planarization of the β-ionone ring is observed on S 1, which may cause a steric lever effect into the protein pocket, thus assisting photoisomerization catalysis. The reported results constitute a solid reference for further studies aimed to rationalize the effect of the environment on the photochemical reactivity of retinal chromophores.

Inter-helical Hydrogen Bonds Are Essential Elements for Intra-protein Signal Transduction: The Role of Asp115 in Bacteriorhodopsin Transport Function

The behavior of the D115A mutant was analyzed by time-resolved UV-Vis and Fourier transformed infrared (FTIR) spectroscopies, aiming to clarify the role of Asp115 in the intra-protein signal transductions occurring during the bacteriorhodopsin photocycle. UV-Vis data on the D115A mutant show severely desynchronized photocycle kinetics. FTIR data show a poor transmission of the retinal isomerization to the chromoprotein, evidenced by strongly attenuated helical changes (amide I), the remarkable absence of environment alterations and protonation/deprotonation events related to Asp96 and direct Schiff base (SB) protonation form the bulk. This argues for the interactions of Asp115 with Leu87 (via water molecule) and Thr90 as key elements for the effective and vectorial proton path between Asp96 and the SB, in the cytoplasmic half of bacteriorhodopsin. The results strongly suggest the presence of a regulation motif enclosed in helices C and D (Thr90–Pro91/Asp115) which drives properly the dynamics of helix C through a set of interactions. It also supports the idea that intra-helical hydrogen bonding clusters in the buried regions of transmembrane proteins can be potential elements in intra-protein signal transduction.

Complex excited dynamics around a plateau on a retinal-like potential surface: chaos, multi-exponential decays and quantum/classical differences

We investigate the classical and quantum dynamics on the plateau of an excited potential energy surface (PES) whose shape mimics the PES driving the photoisomerization of the protonated Schiff base of retinal (PSBR). We adopt a two-dimensional analytical model of the PES, and perform an extended study by varying the potential parameters, revealing a scenario whose interest goes beyond the relevance for the specific case of PSBR. In fact, we document cases with net differences among classical and quantum dynamical predictions, for barrierless PESs. Classical trajectories released on the PES display the signature of chaos and partial trapping on the plateau, whose origin is purely dynamical, since no barrier exists. At variance, on the same barrierless PESs, quantum dynamics does not predict any trapping, always showing a complete depletion of the excited population according to an approximate mono-exponential law. The plateau on the PES promotes complex and unusual dynamical features, and it is sufficient to introduce a very small barrier along the cis–trans torsional mode to give rise to a multi-exponential decay, also at quantum level. Our results are of general interest because plateaux are often found in the excited states of conjugated chromophores.

근접장 마이크로파 현미경을 이용한 로돕신의 광학적 특성 연구

We report the electro-optical properties of the sensory rhodopsin II using a near-field scanning microwave microscope(NSMM). Rhodopsin was known as a photoreceptor pigment with a retinal as a chromophore via a protonated Schiff base and consists of seven <TEX>${\alpha}-helical$</TEX> transmembrane segments. The sensory rhodopsin II, expressing E. coli UT5600 with endogenous retinal biosynthesis system and purified with <TEX>$Ni^{-2}-NTA$</TEX> affinity chromatography in the presence of 0.02 % DM (Dodecyl Maltoside) from Natronomonas pharaonis. We measured the absorption spectra and the transients difference of sensory rhodopsin II from Natronomonas pharaonis using a UV/VIS spectrophotometer with Nd-Yag Laser (532 nm). The absorption spectra of NpSR II showed a typical rhodopsin spectrum with a left shoulder region and the photointermediates spectra of NpSR II-ground state (<TEX>${\lambda}max=498\;nm$</TEX>), NpSR II-M state (<TEX>${\lambda}max=390\;nm$</TEX>), and NpSR II-O state (<TEX>${\lambda}max=550\;nm$</TEX>) during the photocycle. The observed photocycle reaction was confirmed by measuring the microwave reflection coefficient <TEX>$S_{11}$</TEX> at an operating frequency of f=3.93-3.95 GHz and compared with the results of a photocycle of NpSR II.

Mechanism of G‐protein Activation by Rhodopsin†

Rhodopsin is a member of the family of G-protein-coupled receptors (GPCRs), and is an excellent molecular switch for converting light signals into electrical response of the rod photoreceptor cells. Light initiates cis-trans isomerization of the retinal chromophore of rhodopsin and leads to the formation of several thermolabile intermediates during the bleaching process. Recent investigations have identified spectrally distinguishable two intermediate states that can interact with the retinal G-protein, transducin, and have elucidated the functional sharing of these intermediates. The initial contact with GDP-bound G-protein occurs in the meta-Ib intermediate state, which has a protonated Schiff base as its chromophore. The meta-Ib intermediate in the complex with the G-protein converts to the meta-II intermediate with releasing GDP from the alpha-subunit of the G protein. Meta-II has a de-protonated Schiff base chromophore and induces binding of GTP to the alpha-subunit of the G-protein. Thus, the GDP-GTP exchange reaction, namely G-protein activation, by rhodopsin proceeds through at least two steps, with conformational changes in both rhodopsin and the G-protein.