Protonated Retinylidene Schiff Base Research Articles

A single point mutation converts a proton-pumping rhodopsin into a red-shifted, turn-on fluorescent sensor for chloride.

The visualization of chloride in living cells with fluorescent sensors is linked to our ability to design hosts that can overcome the energetic penalty of desolvation to bind chloride in water. Fluorescent proteins can be used as biological supramolecular hosts to address this fundamental challenge. Here, we showcase the power of protein engineering to convert the fluorescent proton-pumping rhodopsin GR from Gloeobacter violaceus into GR1, a red-shifted, turn-on fluorescent sensor for chloride in detergent micelles and in live Escherichia coli. This non-natural function was unlocked by mutating D121, which serves as the counterion to the protonated retinylidene Schiff base chromophore. Substitution from aspartate to valine at this position (D121V) creates a binding site for chloride. The binding of chloride tunes the pKa of the chromophore towards the protonated, fluorescent state to generate a pH-dependent response. Moreover, ion pumping assays combined with bulk fluorescence and single-cell fluorescence microscopy experiments with E. coli, expressing a GR1 fusion with a cyan fluorescent protein, show that GR1 does not pump ions nor sense membrane potential but instead provides a reversible, ratiometric readout of changes in extracellular chloride at the membrane. This discovery sets the stage to use natural and laboratory-guided evolution to build a family of rhodopsin-based fluorescent chloride sensors with improved properties for cellular applications and learn how proteins can evolve and adapt to bind anions in water.

Role of a Helix B Lysine Residue in the Photoactive Site in Channelrhodopsins

In most studied microbial rhodopsins two conserved carboxylic acid residues (the homologs of Asp-85 and Asp-212 in bacteriorhodopsin) and an arginine residue (the homolog of Arg-82) form a complex counterion to the protonated retinylidene Schiff base, and neutralization of the negatively charged carboxylates causes red shifts of the absorption maximum. In contrast, the corresponding neutralizing mutations in some relatively low-efficiency channelrhodopsins (ChRs) result in blue shifts. These ChRs do not contain a lysine residue in the second helix, conserved in higher efficiency ChRs (Lys-132 in the crystallized ChR chimera). By action spectroscopy of photoinduced channel currents in HEK293 cells and absorption spectroscopy of detergent-purified pigments, we found that in tested ChRs the Lys-132 homolog controls the direction of spectral shifts in the mutants of the photoactive site carboxylic acid residues. Analysis of double mutants shows that red spectral shifts occur when this Lys is present, whether naturally or by mutagenesis, and blue shifts occur when it is replaced with a neutral residue. A neutralizing mutation of the Lys-132 homolog alone caused a red spectral shift in high-efficiency ChRs, whereas its introduction into low-efficiency ChR1 from Chlamydomonas augustae (CaChR1) caused a blue shift. Taking into account that the effective charge of the carboxylic acid residues is a key factor in microbial rhodopsin spectral tuning, these findings suggest that the Lys-132 homolog modulates their pKa values. On the other hand, mutation of the Arg-82 homolog that fulfills this role in bacteriorhodopsin caused minimal spectral changes in the tested ChRs. Titration revealed that the pKa of the Asp-85 homolog in CaChR1 lies in the alkaline region unlike in most studied microbial rhodopsins, but is substantially decreased by introduction of a Lys-132 homolog or neutralizing mutation of the Asp-212 homolog. In the three ChRs tested the Lys-132 homolog also alters channel current kinetics.

Vertebrate Ancient-Long Opsin Has Molecular Properties Intermediate between Those of Vertebrate and Invertebrate Visual Pigments

VA/VAL opsin is one of the four kinds of nonvisual opsins that are closely related to vertebrate visual pigments in the phylogenetic tree of opsins. Previous studies indicated that among these opsins, parapinopsin and pinopsin exhibit molecular properties similar to those of invertebrate bistable visual pigments and vertebrate visual pigments, respectively. Here we show that VA/VAL opsin exhibits molecular properties intermediate between those of parapinopsin and pinopsin. VAL opsin from Xenopus tropicalis was expressed in cultured cells, and the pigment with an absorption maximum at 501 nm was reconstituted by incubation with 11-cis-retinal. Light irradiation of this pigment caused cis-to-trans isomerization of the chromophore to form a state having an absorption maximum in the visible region. This state has the ability to activate Gi and Gt types of G proteins. Therefore, the active state of VAL opsin is a visible light-absorbing intermediate, which probably has a protonated retinylidene Schiff base as its chromophore, like the active state of parapinopsin. However, this state was apparently photoinsensitive and did not show reverse reaction to the original pigment, unlike the active state of parapinopsin, and instead similar to that of pinopsin. Furthermore, the Gi activation efficiency of VAL opsin was between those of pinopsin and parapinopsin. Thus, the molecular properties of VA/VAL opsin give insights into the mechanism of conversion of the molecular properties from invertebrate to vertebrate visual pigments.

Structural Changes of Sensory Rhodopsin I and Its Transducer Protein Are Dependent on the Protonated State of Asp76

Sensory rhodopsin I (SRI) functions in both positive and negative phototaxis in complex with halobacterial transducer protein I (HtrI). Orange light activation of SRI results in deprotonation of the retinylidene chromophore of SRI to produce the S 373 photocycle intermediate, the signaling state for positive phototaxis. In this study, we observed pH dependence on structural coupling between the two molecules upon the formation of the S 373 intermediate by means of Fourier transform infrared spectroscopy. At alkaline pH, where Asp76 (one of the counterions of the protonated retinylidene Schiff base) is deprotonated, HtrI-dependent alteration of the light-induced difference spectra is limited to reduction of amide I bands at 1661 (+)/ 1647 (-) cm (-1), and perturbation of one of the protonated carboxylic acid bands occurs at 1734 (-) cm (-1) (which appears to become ionized only when complexed with HtrI). However, at acidic pH, HtrI-complexed SRI exhibits not only light-induced reduction of the amide I changes but a wider range of spectral alterations including the appearance of several new amide I bands, perturbation of the chromophore-related vibrational modes, and other additional changes characteristic of tyrosine, glutamate, and aspartate residues. Since such pH dependence of structural changes was not observed in the complex of the D76N mutant of SRI, which behaves much like HtrI-complexed SRI in acidic conditions, we conclude that extensive orange light-induced conformational coupling between SRI and HtrI occurs only when Asp76 is neutralized.

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.

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.

Highly conserved glutamic acid in the extracellular IV-V loop in rhodopsins acts as the counterion in retinochrome, a member of the rhodopsin family.

Retinochrome is a member of the rhodopsin family having a chromophore retinal and functioning as a retinal photoisomerase in squid photoreceptor cells. Unlike vertebrate rhodopsins, but like many invertebrate rhodopsins, retinochrome does not have a glutamic acid at position 113 to serve as a counterion for the protonated retinylidene Schiff base. Here we investigated possible counterions in retinochrome by site-specific mutagenesis. Our results showed that the counterion is the glutamic acid at position 181, at which almost all the pigments in the rhodopsin family, including vertebrate and invertebrate rhodopsins, have a glutamic or aspartic acid. The remarkable exceptions are the long-wavelength visual pigments that have a histidine that, together with a nearby lysine, serves as a chloride-binding site. Replacement of Glu-181 of bovine rhodopsin with Gln caused a 10-nm red-shift of absorption maximum. Because the position at 181 is in the extracellular loop connecting the transmembrane helices VI and V, these results demonstrate the importance of this loop to function for spectral tuning in the rhodopsin family.

Magic angle spinning NMR of the protonated retinylidene Schiff base nitrogen in rhodopsin: expression of 15N-lysine- and 13C-glycine-labeled opsin in a stable cell line.

The apoprotein corresponding to the mammalian photoreceptor rhodopsin has been expressed by using suspension cultures of HEK293S cells in defined media that contained 6-15N-lysine and 2-13C-glycine. Typical yields were 1.5-1.8 mg/liter. Incorporation of 6-15N-lysine was quantitative, whereas that of 2-13C-glycine was about 60%. The rhodopsin pigment formed by binding of 11-cis retinal was spectrally indistinguishable from native bovine rhodopsin. Magic angle spinning (MAS) NMR spectra of labeled rhodopsin were obtained after its incorporation into liposomes. The 15N resonance corresponding to the protonated retinylidene Schiff base nitrogen was observed at 156.8 ppm in the MAS spectrum of 6-15N-lysine-labeled rhodopsin. This chemical shift corresponds to an effective Schiff base-counterion distance of greater than 4 A, consistent with structural water in the binding site hydrogen bonded with the Schiff base nitrogen and the Glu-113 counterion. The present study demonstrates that structural studies of rhodopsin and other G protein-coupled receptors by using MAS NMR are feasible.

Presence of two rhodopsin intermediates responsible for transducin activation.

To identify how many rhodopsin intermediates interact with retinal G-protein transducin, the photobleaching process of chicken rhodopsin has been investigated in the presence or absence of transducin by means of time-resolved low-temperature spectroscopy. Singular value decomposition (SVD) analysis of the spectral data showed that a new intermediate called meta Ib is present between formally identified metarhodopsin I (now referred to as meta Ia) and metarhodopsin II (meta II). Since the absorption maximum of meta Ib (460 nm) is similar to that of meta Ia (480 nm), but considerably different from that of meta II (380 nm), meta Ib should have a protonated retinylidene Schiff base as its chromophore. Whereas transducin showed no effect on the conversion process between lumirhodopsin (lumi) and meta Ia, it affected the process between meta Ia and meta Ib and that between meta Ib and meta II. These results suggest that at least two intermediates (meta Ib and meta II) interact with transducin. The addition of GTPgammaS had no effect on the meta Ib-transducin interaction, while it abolished the ability of transducin to interact with meta II. Thus, meta Ib only binds to transducin, while meta II catalyzes a GDP-GTP exchange in transducin. These results suggest that deprotonation of the Schiff base chromophore is not necessary for the binding to transducin, while changes in protein structure including Schiff base deprotonation are needed to induce the GDP-GTP exchange in transducin.

Application of NMR‐Spectroscopy to Retinal Proteins

AbstractThe application of NMR spectroscopy to the retinal proteins bacteriorhodopsin (BR) and rhodopsin is reviewed. 2H‐, 15N‐, and 13C‐solid‐state NMR spectroscopy have contributed considerably to understanding the conformation and chemical environment of the protonated retinylidene Schiff base in BR as well as in rhodospin. The data from both pigments clarified the mechanism of the opsin shift which is quite different for rhodopsin and BR. An analysis of the chemical shifts of isotopically labeled aspartic acid, tyrosine, and proline incorporated into BR‐provided evidence for the protonation state of Asp and Tyr, and the isomerization state of the Xaa—Pro peptide bond, respectively. Solid‐state NMR spectroscopy was also applied to the investigation of the photocycle intermediates of BR, as well as bathorhodopsin and metarhodopsin II, which are formed after light‐activation of rhodopsin. Solution NMR spectroscopy of BR solubilized in detergents or organic solvents, as well as of opsin‐derived peptide segments, was also applied to the investigation of the two‐ and three‐dimensional structure of BR.

Rhodopsin activation: effects on the metarhodopsin I-metarhodopsin II equilibrium of neutralization or introduction of charged amino acids within putative transmembrane segments.

We have studied the metarhodopsin I (M I)-metarhodopsin II (M II) equilibria of expressed wild-type and mutant rhodopsins. We studied two classes of mutants with amino acid substitutions in or near the putative transmembrane segments: those in which a charged residue was replaced by a neutral residue (or in one case another charged residue) and those in which a neutral residue likely (or postulated) to be in proximity to the retinylidene Schiff's base was replaced by a charged residue. In the first class, we found mutants that abnormally favored M II (replacements of Asp-83, Glu-134, or Arg-135) as well as one that abnormally favored M I (replacement of Glu-122). In the second class, we found several mutants that abnormally favored M I, the most extreme being those in which glutamate replaced His-211 or Ala-292. These studies suggest that electrostatic forces play a major role in the energetics of the M 1-to-M II transition, and they indicate that electrostatic perturbation in the vicinity of the protonated retinylidene Schiff's base is a plausible mechanism for the change in its pKa that is associated with the M I-M II transition. They further suggest that the highly conserved pair of charged residues homologous to Glu-134 and Arg-135 may play a general role in agonist-dependent conformational changes in G-protein-coupled receptors.

Fourier-transform infrared spectroscopy applied to rhodopsin. The problem of the protonation state of the retinylidene Schiff base re-investigated.

By measuring the rhodopsin--bathorhodopsin, isorhodopsin--bathorhodopsin, rhodopsin--isorhodopsin and rhodopsin--meta-II difference spectra with the method of Fourier-transform infrared spectroscopy we have identified the C = N stretching vibration of the protonated retinylidene Schiff base of rhodopsin, isorhodopsin and bathorhodopsin. In contrast to resonance Raman spectroscopy additional strong bands were observed between 1700 cm-1 and 1620 cm-1. Most of them depend on the isomeric state of the chromophore. The origin of these bands will be discussed. In the fingerprint region isorhodopsin and bathorhodopsin are quite similar but no similarities with infrared spectra of model compounds of any isomeric composition are observed. Therefore, no conclusions on the isomeric state of the retinal in bathorhodopsin can be drawn. We provide evidence for the modification of one or two carboxylic group(s) during the rhodopsin--bathorhodopsin and isorhodopsin--bathorhodopsin transition.

Investigation of the primary photochemistry of bacteriorhodopsin by low-temperature Fourier-transform infrared spectroscopy.

The method of Fourier-transform infrared difference spectroscopy was applied to investigate the transition at 77K of bacteriorhodopsin in its light-adapted form to K6(10), the first intermediate which is stable at low temperature. In addition to unmodified bacteriorhodopsin, bacteriorhodopsin in 2H2O and bacteriorhodopsin containing [15-2H]retinal was used. The results show that major rearrangements occur in the Schiff base in this transition. It is not possible to identify a C = N stretching vibration of the Schiff base in K6(10). The identification of an N-H bending vibration in K6(10) shows that the nitrogen of the previous Schiff base still has a proton attached. The fingerprint region exhibits very unusual features for K6(10) and bears no similarity to protonated retinylidene Schiff base model compounds of any isomeric composition. Therefore, no conclusions on the isomeric state of the retinal in K6(10) can be drawn. The spectra show that the terminal part of the retinal is predominantly reflected in the difference spectra. This indicates that the most polar part of the retinal is located near the Schiff base. We have evidence for protein molecular changes occurring in this transition at 77K.