Factors affecting the C = N stretching in protonated retinal Schiff base: a model study for bacteriorhodopsin and visual pigments

Factors affecting the C = N stretching frequency of protonated retinal Schiff base (RSBH+) were studied with a series of synthetic chromophores and measured under different conditions. Interaction of RSBH+ with nonconjugated positive charges in the vicinity of the ring moiety or a planar polyene conformation (in contrast to the twisted retinal conformation in solution) shifted the absorption maxima but did not affect the C = N stretching frequency. The latter, however, was affected by environmental perturbations in the vicinity of the Schiff base linkage. Diminished ion pairing (i.e., of the positively charged nitrogen to its anion) achieved either by substituting a more bulky counteranion or by designing models with a homoconjugation effect lowered the C = N stretch energy. Decreasing solvation of the positively charged nitrogen leads to a similar trend. These effects in the vicinity of the Schiff base linkage also perturb the deuterium isotope effect observed upon deuteriation of the Schiff base. The results are interpreted by considering the mixing of the C = N stretching and C = N-H bending vibration. The C = N mode is shifted due to electrostatic interaction with nonconjugated positive charges in the vicinity of the Schiff base linkage, an interaction that does not influence the isotope effect. Weak hydrogen bonding between the Schiff base linkage in bacteriorhodopsin (bR) and its counteranion or, alternatively, poor solvation of the positively charged Schiff base nitrogen can account for the C = N stretching frequency of 1640 cm-1 and the deuterium isotope effect of 17 cm-1 observed in this pigment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Light activation of the isomerization and deprotonation of the protonated Schiff base retinal

We perform an ab initio analysis of the photoisomerization of the protonated Schiff base of retinal (PSB-retinal) from 11-cis to 11-trans rotating the C10-C11=C12-C13 dihedral angle from 0° (cis) to -180° (trans). We find that the retinal molecule shows the lowest rotational barrier (0.22 eV) when its charge state is zero as compared to the barrier for the protonated molecule which is ∼0.89 eV. We conclude that rotation most likely takes place in the excited state of the deprotonated retinal. The addition of a proton creates a much larger barrier implying a switching behavior of retinal that might be useful for several applications in molecular electronics. All conformations of the retinal compound absorb in the green region with small shifts following the dihedral angle rotation; however, the Schiff base of retinal (SB-retinal) at trans-conformation absorbs in the violet region. The rotation of the dihedral angle around the C11=C12 π-bond affects the absorption energy of the retinal and the binding energy of the SB-retinal with the proton at the N-Schiff; the binding energy is slightly lower at the trans-SB-retinal than at other conformations of the retinal.     

Phototransduction by vertebrate ultraviolet visual pigments: protonation of the retinylidene Schiff base following photobleaching

The photochemical and subsequent thermal reactions of the mouse short-wavelength visual pigment (MUV) were studied by using cryogenic UV-visible and FTIR difference spectroscopy. Upon illumination at 75 K, MUV forms a batho intermediate (lambda(max) approximately 380 nm). The batho intermediate thermally decays to the lumi intermediate (lambda(max) approximately 440 nm) via a slightly blue-shifted intermediate not observed in other photobleaching pathways, BL (lambda(max) approximately 375 nm), at temperatures greater than 180 K. The lumi intermediate has a significantly red-shifted absorption maximum at 440 nm, suggesting that the retinylidene Schiff base in this intermediate is protonated. The lumi intermediate decays to an even more red-shifted meta I intermediate (lambda(max) approximately 480 nm) which in turn decays to meta II (lambda(max) approximately 380 nm) at 248 K and above. Differential FTIR analysis of the 1100-1500 cm(-1) region reveals an integral absorptivity that is more than 3 times smaller than observed in rhodopsin and VCOP. These results are consistent with an unprotonated Schiff base chromophore. We conclude that the MUV-visual pigment possesses an unprotonated retinylidene Schiff base in the dark state, and undergoes a protonation event during the photobleaching cascade.     

Regulation of phototransduction in short-wavelength cone visual pigments via the retinylidene Schiff base counterion.

Short-wavelength visual pigments (SWS1) have lambda(max) values that range from the ultraviolet to the blue. Like all visual pigments, this class has an 11-cis-retinal chromophore attached through a Schiff base linkage to a lysine residue of opsin apoprotein. We have characterized a series of site-specific mutants at a conserved acidic residue in transmembrane helix 3 in the Xenopus short-wavelength sensitive cone opsin (VCOP, lambda(max) approximately 427 nm). We report the identification of D108 as the counterion to the protonated retinylidene Schiff base. This residue regulates the pK(a) of the Schiff base and, neutralizing this charge, converts the violet sensitive pigment into one that absorbs maximally in the ultraviolet region. Changes to this position cause the pigment to exhibit two chromophore absorbance bands, a major band with a lambda(max) of approximately 352-372 nm and a minor, broad shoulder centered around 480 nm. The behavior of these two absorbance bands suggests that these represent unprotonated and protonated Schiff base forms of the pigment. The D108A mutant does not activate bovine rod transducin in the dark but has a significantly prolonged lifetime of the active MetaII state. The data suggest that in short-wavelength sensitive cone visual pigments, the counterion is necessary for the characteristic rapid production and decay of the active MetaII state.     

E113 is required for the efficient photoisomerization of the unprotonated chromophore in a UV-absorbing visual pigment

Protonation of the retinal Schiff base chromophore is responsible for the absorption of visible light and is stabilized by the counterion residue E113 in vertebrate visual pigments. However, this residue is also conserved in vertebrate UV-absorbing visual pigments (UV pigments) which have an unprotonated Schiff base chromophore. To elucidate the role played by this residue in the photoisomerization of the unprotonated chromophore in UV pigments, we measured the quantum yield of the E113Q mutant of the mouse UV cone pigment (mouse UV). The quantum yield of the mutant was much lower than that of the wild type, indicating that E113 is required for the efficient photoisomerization of the unprotonated chromophore in mouse UV. Introduction of the E113Q mutation into the chicken violet cone pigment (chicken violet), which has a protonated chromophore, caused deprotonation of the chromophore and a reduction in the quantum yield. On the other hand, the S90C mutation in chicken violet, which deprotonated the chromophore with E113 remaining intact, did not significantly affect the quantum yield. These results suggest that E113 facilitates photoisomerization in both UV-absorbing and visible light-absorbing visual pigments and provide a possible explanation for the complete conservation of E113 among vertebrate UV pigments.     

Regulation of photoactivation in vertebrate short wavelength visual pigments: protonation of the retinylidene Schiff base and a counterion switch

Xenopus violet cone opsin (VCOP) and its counterion variant (VCOP-D108A) are expressed in mammalian COS1 cells and regenerated with 11-cis-retinal. The phototransduction process in VCOP-D108A is investigated via cryogenic electronic spectroscopy, homology modeling, molecular dynamics, and molecular orbital theory. The VCOP-D108A variant is a UV-like pigment that displays less efficient photoactivation than the mouse short wavelength sensitive visual pigment (MUV) and photobleaching properties that are significantly different. Theoretical calculations trace the difference to the protonation state of the nearby glutamic acid residue E176, which is the homology equivalent of E181 in rhodopsin. We find that E176 is negatively charged in MUV but neutral (protonated) in VCOP-D108A. In the dark state, VCOP-D108A has an unprotonated Schiff base (SB) chromophore (lambdamax = 357 nm). Photolysis of VCOP-D108A at 70 K generates a bathochromic photostationary state (lambdamax = 380 nm). We identify two lumi intermediates, wherein the transitions from batho to the lumi intermediates are temperature- and pH-dependent. The batho intermediate decays to a more red-shifted intermediate called lumi I. The SB becomes protonated during the lumi I to lumi II transition. Decay of lumi II forms meta I, followed by the formation of meta II. We conclude that even in the absence of a primary counterion in VCOP-D108A, the SB becomes protonated during the photoactivation cascade. We examine the relevance of this observation to the counterion switch mechanism of visual pigment activation.     

Serine 85 in transmembrane helix 2 of short-wavelength visual pigments interacts with the retinylidene Schiff base counterion.

Short-wavelength cone visual pigments (SWS1) are responsible for detecting light from 350 to 430 nm. Models of this class of pigment suggest that TM2 has extensive contacts with the retinal binding pocket and stabilizes interhelical interactions. The role of TM2 in the structure-function of the Xenopus SWS1 (VCOP, lambda(max) = 427 nm) pigment was studied by replacement of the helix with that of bovine rhodopsin and also by mutagenesis of highly conserved residues. The TM2 chimera and G78D, F79L, M81E, P88T, V89S, and F90V mutants did not produce any significant spectral shift of the dark state or their primary photointermediate formed upon illumination at cryogenic temperatures. The mutant G77R (responsible for human tritanopia) was completely defective in folding, while C82A and F87T bound retinal at reduced levels. The position S85 was crucial for obtaining the appropriate spectroscopic properties of VCOP. S85A and S85T did not bind retinal. S85D bound retinal and had a wild-type dark state at room temperature and a red-shifted dark state at 45 K and formed an altered primary photointermediate. S85C absorbed maximally at 390 nm at neutral pH and at 365 nm at pH >7.5. The S85C dark state was red shifted by 20 nm at 45 K and formed an altered primary photointermediate. These data suggest that S85 is involved in a hydrogen bond with the protonated retinylidene Schiff base counterion in both the dark state and the primary photointermediate.     

Infrared spectroscopic analysis on structural changes around the protonated Schiff base upon retinal isomerization in light-driven sodium pump KR2

Krokinobacter rhodopsin 2 (KR2) was discovered as the first light-driven sodium pumping rhodopsin (NaR) in 2013, which contains unique amino acid residues on C-helix (N112, D116, and Q123), referred to as an NDQ motif. Based on the recent X-ray crystal structures of KR2, the sodium transport pathway has been investigated by various methods. However, due to complicated structural information around the protonated Schiff base (PRSB) region in the dark state and lack of structural information in the intermediates with sodium bound in KR2, detailed sodium pump mechanism is still unclear. Here we applied comprehensive low-temperature light-induced difference FTIR spectroscopy on isotopically labeled KR2 WT and site-directed mutant proteins (N112A, D116E, R109A, and R109K). We assigned the N-D stretching vibration of the PRSB at 2095 cm⁻¹ and elucidate the hydrogen bonding interaction with D116 (a counter ion for the PRSB). We also assigned strongly hydrogen-bonded water (2333 cm⁻¹) near R109 and D251, and found that presence of a positive charge at the position of R109 is prerequisite for the pumping function of KR2.     

Cone visual pigments and retinal mosaics in the striped marlin

Three different cone photoreceptor visual pigments in the retina of striped marlin Tetrapturus audax were found with the aid of microspectrophotometry. This provides the first evidence for the basis of colour vision in the Istiophoridae. Furthermore, regional variations in photoreceptor density, type and spatial arrangement indicate differing visual capabilities along different visual axes.     

Localization of the retinal protonated Schiff base counterion in rhodopsin.

Semiempirical molecular orbital calculations are combined with 13C NMR chemical shifts to localize the counterion in the retinal binding site of vertebrate rhodopsin. Charge densities along the polyene chain are calculated for an 11-cis-retinylidene protonated Schiff base (11-cis-RPSB) chromophore with 1) a chloride counterion at various distances from the Schiff base nitrogen, 2) one or two chloride counterions at different positions along the retinal chain from C10 to C15 and at the Schiff base nitrogen, and 3) a carboxylate counterion out of the retinal plane near C12. Increasing the distance of the negative counterion from the Schiff base results in an enhancement of alternating negative and positive partial charge on the even- and odd-numbered carbons, respectively, when compared to the 11-cis-RPSB chloride model compound. In contrast, the observed 13C NMR data of rhodopsin exhibit downfield chemical shifts from C8 to C13 relative to the 11-cis-RPSB.Cl corresponding to a net increase of partial positive or decrease of partial negative charge at these positions (Smith, S. O., I. Palings, M. E. Miley, J. Courtin, H. de Groot, J. Lugtenburg, R. A. Mathies, and R. G. Griffin. 1990. Biochemistry. 29:8158-8164). The anomalous changes in charge density reflected in the rhodopsin NMR chemical shifts can be qualitatively modeled by placing a single negative charge above C12. The calculated fit improves when a carboxylate counterion is used to model the retinal binding site. Inclusion of water in the model does not alter the fit to the NMR data, although it is consistent with observations based on other methods.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein changes associated with reprotonation of the Schiff base in the photocycle of Asp96-->Asn bacteriorhodopsin. The MN intermediate with unprotonated Schiff base but N-like protein structure.

The difference Fourier transform infrared spectrum for the N intermediate in the photoreaction of the light-adapted form of bacteriorhodopsin can be recorded at pH 10 at 274 K (Pfefferlé, J.-M., Maeda, A., Sasaki, J., and Yoshizawa, T. (1991) Biochemistry 30, 6548-6556). Under these conditions, Asp96-->Asn bacteriorhodopsin gives a photoproduct which shows changes in protein structure similar to those observed in N of wild-type bacteriorhodopsin. However, decreased intensity of the chromophore bands and the single absorbance maximum at about 400 nm indicate that the Schiff base is unprotonated, as in the M intermediate. This photoproduct was named MN. At pH 7, where the supply of proton is not as restricted as at pH 10, Asp96-->Asn bacteriorhodopsin yields N with a protonated Schiff base. The Asn96 residue, which cannot deprotonate as Asp96 in wild-type bacteriorhodopsin, is perturbed upon formation of both MN at pH 10 and N at pH 7. We suggest that the reprotonation of the Schiff base is preceded by a large change in the protein structure including perturbation of the residue at position 96.     

Vibrational analysis of the all-trans retinal protonated Schiff base.

We have obtained Raman spectra of a series of all-trans retinal protonated Schiff-base isotopic derivatives. 13C-substitutions were made at the 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 positions while deuteration was performed at position 15. Based on the isotopic shifts, the observed C--C stretching vibrations in the 1,100-1,400 cm-1 fingerprint region are assigned. Normal mode calculations using a modified Urey-Bradley force field have been refined to reproduce the observed frequencies and isotopic shifts. Comparison with fingerprint assignments of all-trans retinal and its unprotonated Schiff base shows that the major effect of Schiff-base formation is a shift of the C14--C15 stretch from 1,111 cm-1 in the aldehyde to approximately 1,163 cm-1 in the Shiff base. This shift is attributed to the increased C14--C15 bond order that results from the reduced electronegativity of the Schiff-base nitrogen compared with the aldehyde oxygen. Protonation of the Schiff base increases pi-electron delocalization, causing a 6 to 16 cm-1 frequency increase of the normal modes involving the C8--C9, C10--C11, C12--C13, and C14--C15 stretches. Comparison of the protonated Schiff base Raman spectrum with that of light-adapted bacteriorhodopsin (BR568) shows that incorporation of the all-trans protonated Schiff base into bacterio-opsin produces an additional approximately 10 cm-1 increase of each C--C stretching frequency as a result of protein-induced pi-electron delocalization. Importantly, the frequency ordering and spacing of the C--C stretches in BR568 is the same as that found in the protonated Schiff base.     

Role of the 9-methyl group of retinal in cone visual pigments

In rhodopsin, the 9-methyl group of retinal has previously been identified as being critical in linking the ligand isomerization with the subsequent protein conformational changes that result in the activation of its G protein, transducin. Here, we report studies on the role of this methyl group in the salamander rod and cone pigments. Pigments were generated by combining proteins expressed in COS cells with 11-cis 9-demethyl retinal, where the 9-methyl group on the polyene chain has been deleted. The absorption spectra of all pigments were blue-shifted. The red cone and blue cone/green rod pigments were unstable to hydroxylamine; whereas, the rhodopsin and UV cone pigments were stable. The lack of the 9-methyl group of the chromophore did not affect the ability of the red cone and blue cone/green rod pigments to activate transducin. On the other hand, with the rhodopsin and UV cone pigments, activation was diminished. Interestingly, the red cone pigment containing the retinal analogue remained active longer than the native pigment. Thus, the 9-methyl group of retinal is not important in the activation pathway of the red cone and blue cone/green rod pigments. However, for the red cone pigment, the 9-methyl group of retinal appears to be critical in the deactivation pathway.     

Excited-state dynamics of all-trans protonated retinal Schiff base in CRABPII-based rhodopsin mimics

The rhodopsin mimic is a chemically synthetized complex with retinyl Schiff base (RSB) formed between protein and the retinal chromophore that can mimic the natural rhodopsin-like protein. The artificial rhodopsin mimic is more stable and designable than the natural protein and hence has wider uses in photon detection devices. The mimic structure RSB, like the case in the actual rhodopsin-like protein, undergoes isomerization and protonation throughout the photoreaction process. As a result, understanding the dynamics of the RSB in the photoreaction process is critical. In this study, the ultrafast transient absorption spectra of three mutants of the cellular retinoic acid-binding protein II-based rhodopsin mimic at acidic environment were recorded, from which the related excited-state dynamics of the all-trans protonated RSB (AT-PRSB) were investigated. The transient fluorescence spectra measurements are used to validate some of the dynamic features. We find that the excited-state dynamics of AT-PRSB in three mutants share a similar pattern that differs significantly from the dynamics of 15-cis PRSB of the rhodopsin mimic in neutral solution. By comparing the dynamics across the three mutants, we discovered that the aromatic residues near the β-ionone ring structure of the retinal may help stabilize the AT-PRSB and hence slow down its isomerization rate. The experimental results provide implications on designing a rhodopsin-like protein with significant infrared fluorescence, which can be particularly useful in the applications in biosensing or bioimaging in deeper tissues.     

Ultrafast excited state dynamics of the protonated Schiff base of all-trans retinal in solvents

We present a comparative study of the ultrafast photophysics of all-trans retinal in the protonated Schiff base form in solvents with different polarities and viscosities. Steady-state spectra of retinal in the protonated Schiff base form show large absorption-emission Stokes shifts (6500-8100 cm(-1)) for both polar and nonpolar solvents. Using a broadband fluorescence up-conversion experiment, the relaxation kinetics of fluorescence is investigated with 120 fs time resolution. The time-zero spectra already exhibit a Stokes-shift of approximately 6000 cm(-1), indicating depopulation of the Franck-Condon region in < or =100 fs. We attribute it to relaxation along skeletal stretching. A dramatic spectral narrowing is observed on a 150 fs timescale, which we assign to relaxation from the S(2) to the S(1) state. Along with the direct excitation of S(1), this relaxation populates different quasistationary states in S(1), as suggested from the existence of three distinct fluorescence decay times with different decay associated spectra. A 0.5-0.65 ps decay component is observed, which may reflect the direct repopulation of the ground state, in line with the small isomerization yield in solvents. Two longer decay components are observed and are attributed to torsional motion leading to photo-isomerization. The various decay channels show little or no dependence with respect to the viscosity or dielectric constant of the solvents. This suggests that in the protein, the bond selectivity of isomerization is mainly governed by steric effects.     

The pKa of the protonated Schiff bases of gecko cone and octopus visual pigments.

A visual pigment is composed of retinal bound to its apoprotein by a protonated Schiff base linkage. Light isomerizes the chromophore and eventually causes the deprotonation of this Schiff base linkage at the meta II stage of the bleaching cycle. The meta II intermediate of the visual pigment is the active form of the pigment that binds to and activates the G protein transducin, starting the visual cascade. The deprotonation of the Schiff base is mandatory for the formation of meta II intermediate. We studied the proton binding affinity, pKa, of the Schiff base of both octopus rhodopsin and the gecko cone pigment P521 by spectral titration. Several fluorinated retinal analogs have strong electron withdrawing character around the Schiff base region and lower the Schiff base pKa in model compounds. We regenerated octopus and gecko visual pigments with these fluorinated and other retinal analogs. Experiments on these artificial pigments showed that the spectral changes seen upon raising the pH indeed reflected the pKa of the Schiff base and not the denaturation of the pigment or the deprotonation of some other group in the pigment. The Schiff base pKa is 10.4 for octopus rhodopsin and 9.9 for the gecko cone pigment. We also showed that although the removal of Cl- ions causes considerable blue-shift in the gecko cone pigment P521, it affects the Schiff base pKa very little, indicating that the lambda max of visual pigment and its Schiff base pKa are not tightly coupled.     

Role of the retinal hydrogen bond network in rhodopsin Schiff base stability and hydrolysis

Little is known about the molecular mechanism of Schiff base hydrolysis in rhodopsin. We report here our investigation into this process focusing on the role of amino acids involved in a hydrogen bond network around the retinal Schiff base. We find conservative mutations in this network (T94I, E113Q, S186A, E181Q, Y192F, and Y268F) increase the activation energy (E(a)) and abolish the concave Arrhenius plot normally seen for Schiff base hydrolysis in dark state rhodopsin. Interestingly, two mutants (T94I and E113Q) show dramatically faster rates of Schiff base hydrolysis in dark state rhodopsin, yet slower hydrolysis rates in the active MII form. We find deuterium affects the hydrolysis process in wild-type rhodopsin, exhibiting a specific isotope effect of approximately 2.5, and proton inventory studies indicate that multiple proton transfer events occur during the process of Schiff base hydrolysis for both dark state and MII forms. Taken together, our study demonstrates the importance of the retinal hydrogen bond network both in maintaining Schiff base integrity in dark state rhodopsin, as well as in catalyzing the hydrolysis and release of retinal from the MII form. Finally, we note that the dramatic alteration of Schiff base stability caused by mutation T94I may play a causative role in congenital night blindness as has been suggested by the Oprian and Garriga laboratories.     

Longitudinal leaf gradients of UV-absorbing screening pigments in barley (Hordeum vulgare)

In vivo leaf characteristics were examined to describe longitudinal gradients of UV-absorbing screening pigments in barley. Chlorophyll fluorescence properties and in vivo absorption spectra (210–750 nm) of leaves were measured from the base to the tip. Barley leaves showed strong longitudinal gradients of chlorophyll, where chlorophyll concentration increased within the first 5–8 cm from the leaf base, and did not significantly change for the remaining part of the leaf. Fluorescence microscopy was used to localize cell wall bound screening pigments different from flavonoids, since flavonoids lack a blue-green fluorescence emission (Lichtenthaler and Schweiger 1998). Measurements of in vivo chlorophyll fluorescence indicated that the ratio of UV-absorbing screening pigments per leaf area increases from the leaf base to the tip. These gradients were confirmed by in vivo absorption spectra. It is demonstrated that leaves in the early stage of development are less protected against UV-radiation than fully developed mature leaf regions. The experiments show that measurements of in vivo chlorophyll fluorescence are ideally suited as a fast non-invasive tool to estimate the epidermal UV-transmittance in different leaf sections.