Light- and temperature-dependent dynamics of chromophore and protein structural changes in bathy phytochrome Agp2.

Bacterial phytochromes are sensoric photoreceptors that transform light absorbed by the photosensor core module (PCM) to protein structural changes that eventually lead to the activation of the enzymatic output module. The underlying photoinduced reaction cascade in the PCM starts with the isomerization of the tetrapyrrole chromophore, followed by conformational relaxations, proton transfer steps, and a secondary structure transition of a peptide segment (tongue) that is essential for communicating the signal to the output module. In this work, we employed various static and time-resolved IR and resonance Raman spectroscopic techniques to study the structural and reaction dynamics of the Meta-F intermediate of both the PCM and the full-length (PCM and output module) variant of the bathy phytochrome Agp2 from Agrobacterium fabrum. In both cases, this intermediate represents a branching point of the phototransformation, since it opens an unproductive reaction channel back to the initial state and a productive pathway to the final active state, including the functional protein structural changes. It is shown that the functional quantum yield, i.e. the events of tongue refolding per absorbed photons, is lower by a factor of ca. two than the quantum yield of the primary photochemical process. However, the kinetic data derived from the spectroscopic experiments imply an increased formation of the final active state upon increasing photon flux or elevated temperature under photostationary conditions. Accordingly, the branching mechanism does not only account for the phytochrome's function as a light intensity sensor but may also modulate its temperature sensitivity.

Local Electric Field Changes during the Photoconversion of the Bathy Phytochrome Agp2.

Phytochromes switch between a physiologically inactive and active state via a light-induced reaction cascade, which is initiated by isomerization of the tetrapyrrole chromophore and leads to the functionally relevant secondary structure transition of a protein segment (tongue). Although details of the underlying cause-effect relationships are not known, electrostatic fields are likely to play a crucial role in coupling chromophores and protein structural changes. Here, we studied local electric field changes during the photoconversion of the dark state Pfr to the photoactivated state Pr of the bathy phytochrome Agp2. Substituting Tyr165 and Phe192 in the chromophore pocket by para-cyanophenylalanine (pCNF), we monitored the respective nitrile stretching modes in the various states of photoconversion (vibrational Stark effect). Resonance Raman and IR spectroscopic analyses revealed that both pCNF-substituted variants undergo the same photoinduced structural changes as wild-type Agp2. Based on a structural model for the Pfr state of F192pCNF, a molecular mechanical-quantum mechanical approach was employed to calculate the electric field at the nitrile group and the respective stretching frequency, in excellent agreement with the experiment. These calculations serve as a reference for determining the electric field changes in the photoinduced states of F192pCNF. Unlike F192pCNF, the nitrile group in Y165pCNF is strongly hydrogen bonded such that the theoretical approach is not applicable. However, in both variants, the largest changes of the nitrile stretching modes occur in the last step of the photoconversion, supporting the view that the proton-coupled restructuring of the tongue is accompanied by a change of the electric field.

Intramolecular Proton Transfer Controls Protein Structural Changes in Phytochrome.

Phytochromes are biological photoswitches that interconvert between two parent states (Pr and Pfr). The transformation is initiated by photoisomerization of the tetrapyrrole chromophore, followed by a sequence of chromophore and protein structural changes. In the last step, a phytochrome-specific peptide segment (tongue) undergoes a secondary structure change, which in prokaryotic phytochromes is associated with the (de)activation of the output module. The focus of this work is the Pfr-to-Pr photoconversion of the bathy bacteriophytochrome Agp2 in which Pfr is the thermodynamically stable state. Using spectroscopic techniques, we studied the structural and functional consequences of substituting Arg211, Tyr165, His278, and Phe192 close to the biliverdin (BV) chromophore. In Pfr, substitutions of these residues do not affect the BV structure. The characteristic Pfr properties of bathy phytochromes, including the protonated propionic side chain of ring C (propC) of BV, are preserved. However, replacing Arg211 or Tyr165 blocks the photoconversion in the Meta-F state, prior to the secondary structure transition of the tongue and without deprotonation of propC. The Meta-F state of these variants displays low photochemical activity, but electronic excitation causes ultrafast alterations of the hydrogen bond network surrounding the chromophore. In all variants studied here, thermal back conversion from the photoproducts to Pfr is decelerated but substitution of His278 or Phe192 is not critical for the Pfr-to-Pr photoconversion. These variants do not impair deprotonation of propC or the α-helix/ß-sheet transformation of the tongue during the Meta-F-to-Pr decay. Thus, we conclude that propC deprotonation is essential for restructuring of the tongue.

Role of the Propionic Side Chains for the Photoconversion of Bacterial Phytochromes.

Bacteriophytochromes harboring a biliverdin IXα (BV) chromophore undergo photoinduced reaction cascades to switch between physiologically inactive and active states. Employing vibrational spectroscopic and computational methods, we analyzed the role of propionic substituents of BV in the transformations between parent states Pr and Pfr in prototypical (Agp1) and bathy (Agp2) phytochromes from Agrobacterium fabrum. Both proteins form adducts with BV monoesters (BVM), esterified at propionic side chain B (PsB) or C (PsC), but in each case, only one monoester adduct is reactive. In the reactive Agp2-BVM-B complex (esterified at ring B), the Pfr dark state displays the structural properties characteristic of bathy phytochromes, including a protonated PsC. As in native Agp2, PsC is deprotonated in the final step of the Pfr phototransformation. However, the concomitant α-helix/ß-sheet secondary structure change of the tongue is blocked at the stage of unfolding of the coiled loop region. This finding and the shift of the tautomeric equilibrium of BVM toward the enol form are attributed to the drastic changes in the electrostatic potential. The calculations further suggest that deprotonation of PsC and the protonation state of His278 control the reactivity of the enol tautomer, thereby accounting for the extraordinarily slow thermal reversion. Although strong perturbations of the electrostatic potential are also found for Agp1-BVM, the consequences for the Pr-to-Pfr phototransformation are less severe. Specifically, the structural transition of the tongue is not impaired and thermal reversion is even accelerated. The different response of Agp1 and Agp2 to monoesterification of BV points to different photoconversion mechanisms.

Photoreactions of the Histidine Kinase Rhodopsin Ot-HKR from the Marine Picoalga Ostreococcus tauri.

The tiny picoalga, Ostreococcus tauri, originating from the Thau Lagoon is a member of the marine phytoplankton. Because of its highly reduced genome and small cell size, while retaining the fundamental requirements of a eukaryotic photosynthetic cell, it became a popular model organism for studying photosynthesis or circadian clock-related processes. We analyzed the spectroscopic properties of the photoreceptor domain of the histidine kinase rhodopsin Ot-HKR that is suggested to be involved in the light-induced entrainment of the Ostreococcus circadian clock. We found that the rhodopsin, Ot-Rh, dark state absorbs maximally at 505 nm. Exposure to green-orange light led to the accumulation of a blue-shifted M-state-like absorbance form with a deprotonated Schiff base. This Ot-Rh P400 state had an unusually long lifetime of several minutes. A second long-living photoproduct with a red-shifted absorbance, P560, accumulated upon illumination with blue/UVA light. The resulting photochromicity of the rhodopsin is expected to be advantageous to its function as a molecular control element of the signal transducing HKR domains. The light intensity and the ratio of blue vs green light are reflected by the ratio of rhodopsin molecules in the long-living absorbance forms. Furthermore, dark-state absorbance and the photocycle kinetics vary with the salt content of the environment substantially. This observation is attributed to anion binding in the dark state and a transient anion release during the photocycle, indicating that the salinity affects the photoinduced processes.

Microbial Halorhodopsins: Light-Driven Chloride Pumps.

Early research on the four microbial rhodopsins discovered in the archaeal Halobacterium salinarum revealed a structural template that served as a scaffold for two different functions: light-driven ion transport and phototaxis. Bacteriorhodopsin and halorhodopsin are proton and chloride pumps, respectively, while sensory rhodopsin I and II are responsible for phototactic behavior of the archaea. Halorhodopsins have been identified in various other species. Besides this group of archaeal halorhodopsins distinct chloride transporting rhodopsins groups have recently been identified in other organism like Flavobacteria or Cyanobacteria. Halorhodopsin from Natronomonas pharaonis is the best-studied homologue because of its facile expression and purification and its advantageous properties, which was the reason to introduce this protein as neural silencer into the new field of optogenetics. Two other major families of genetically encoded silencing proteins, proton pumps and anion channels, extended the repertoire of optogenetic tools. Here, we describe the functional and structural characteristics of halorhodopsins. We will discuss the data in light of common principles underlying the mechanism of ion pumps and sensors and will review biophysical and biochemical aspects of neuronal silencers.

Common Structural Elements in the Chromophore Binding Pocket of the Pfr State of Bathy Phytochromes.

Phytochromes are bimodal photoreceptors which, upon light absorption by the tetrapyrrole chromophore, can be converted between a red-absorbing state (Pr) and far-red-absorbing state (Pfr). In bacterial phytochromes, either Pr or Pfr are the thermally stable states, thereby constituting the classes of prototypical and bathy phytochromes, respectively. In this work, we have employed vibrational spectroscopies to elucidate the origin of the thermal stability of the Pfr states in bathy phytochromes. Here, we present the first detailed spectroscopic analysis of RpBphP6 (Rhodopseudomas palustris), which together with results obtained for Agp2 (Agrobacterium tumefaciens) and PaBphP (Pseudomonas aeruginosa) allows identifying common structural properties of the Pfr state of bathy phytochromes, which are (1) a homogenous chromophore structure, (2) the protonated ring C propionic side chain of the chromophore and (3) a retarded H/D exchange at the ring D nitrogen. These properties are related to the unique strength of the hydrogen bonding interactions between the ring D N-H group with the side chain of the conserved Asp194 (PaBphP numbering). As revealed by a comparative analysis of homology models and available crystal structures of Pfr states, these interactions are strengthened by an Arg residue (Arg453) only in bathy but not in prototypical phytochromes.

A Red/Green Cyanobacteriochrome Sustains Its Color Despite a Change in the Bilin Chromophore's Protonation State.

The second GAF domain of AnPixJ, AnPixJg2, a bilin-binding protein from the cyanobacterium Anabaena PCC 7120, undergoes a photoinduced interconversion between a red-absorbing state, Pr, and a green-absorbing state, Pg. Combining ultraviolet-vis (UV-vis), infrared, resonance Raman (RR), and magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, we have studied this cyanobacteriochrome (CBCR) assembled with phycocyanobilin (PCB) either in vivo or in vitro. In both assembly routes, the spectroscopic data of the Pr state reveal nearly identical chromophore structures with a protonated (cationic) bilin. However, unlike the native (in vivo assembly) Pg photoproduct, in which the bilin retains protonation, the Pg generated from the in vitro-assembled AnPixJg2 harbors a deprotonated (neutral) bilin chromophore at pH 7.8. IR difference spectroscopy further reveals the transfer of a proton from the bilin to a side-chain carboxylate on an amino acid, probably Asp291. Besides the change in protonation state, the bilin structure is very similar in the in vitro- and in vivo-assembled Pg photoproducts. The chromophore of the in vitro Pg becomes protonated when the pH is increased to 10, presumably because of a partial reversal of protein misfolding. Most remarkably, the electronic transitions remain unchanged and are very similar to those of the native Pg. Thus, bilin protonation is not a key parameter for controlling the energies of the electronic transitions in AnPixJg2. Possible alternative molecular mechanisms for color tuning are discussed.

A protonation-coupled feedback mechanism controls the signalling process in bathy phytochromes.

Phytochromes are bimodal photoswitches composed of a photosensor and an output module. Photoactivation of the sensor is initiated by a double bond isomerization of the tetrapyrrole chromophore and eventually leads to protein conformational changes. Recently determined structural models of phytochromes identify differences between the inactive and the signalling state but do not reveal the mechanism of photosensor activation or deactivation. Here, we report a vibrational spectroscopic study on bathy phytochromes that demonstrates that the formation of the photoactivated state and thus (de)activation of the output module is based on proton translocations in the chromophore pocket coupling chromophore and protein structural changes. These proton transfer steps, involving the tetrapyrrole and a nearby histidine, also enable thermal back-isomerization of the chromophore via keto-enol tautomerization to afford the initial dark state. Thus, the same proton re-arrangements inducing the (de)activation of the output module simultaneously initiate the reversal of this process, corresponding to a negative feedback mechanism.

Conserved Tyr223(5.58) plays different roles in the activation and G-protein interaction of rhodopsin.

Rhodopsin, a seven transmembrane helix (TM) receptor, binds its ligand 11-cis-retinal via a protonated Schiff base. Coupling to the G-protein transducin (G(t)) occurs after light-induced cis/trans-retinal isomerization, which leads through photoproducts into a sequence of metarhodopsin (Meta) states: Meta I ⇌ Meta IIa ⇌ Meta IIb ⇌ Meta IIbH(+). The structural changes behind this three-step activation scheme are mediated by microswitch domains consisting of conserved amino acids. Here we focus on Tyr223(5.58) as part of the Y(5.58)X(7)K(R)(5.66) motif. Mutation to Ala, Phe, or Glu results in specific impairments of G(t)-activation measured by intrinsic G(t) fluorescence. UV-vis/FTIR spectroscopy of rhodopsin and its complex with a C-terminal G(t)α peptide allows the assignment of these deficiencies to specific steps in the activation path. Effects of mutation occur already in Meta I but do not directly influence deprotonation of the Schiff base during formation of Meta IIa. Absence of the whole phenol ring (Y223A) allows the activating motion of TM6 in Meta IIb but impairs the coupling to G(t). When only the hydroxyl group is lacking (Y223F), Meta IIb does not accumulate, but the activity toward G(t) remains substantial. From the FTIR features of Meta IIbH(+) we conclude that proton uptake to Glu134(3.49) is mandatory for Tyr223(5.58) to engage in the interaction with the key player Arg135(3.50) predicted by X-ray analysis. This polar interaction is partially recovered in Y223E, explaining its relatively high activity. Only the phenol side chain of tyrosine provides all characteristics for accumulation of the active state and G-protein activation.

The photoreactions of recombinant phytochrome CphA from the cyanobacterium Calothrix PCC7601: a low-temperature UV-Vis and FTIR study.

The photoreactions of recombinant phytochrome CphA from cyanobacterium Calothrix sp. PCC7601 reconstituted with phycocyanobilin were investigated using UV-Vis and Fourier transform infrared (FTIR) difference spectroscopy, stabilizing intermediates at low temperature. The yield of the forward reaction strongly depends on temperature, unlike the backward reaction. Because of the very fast thermal relaxation processes in the Pr to Pfr pathway, no pure difference spectra of the Pr photoconversion products could be directly measured. Thus, the contribution of the Pfr:Pr pathway was taken into account by applying an appropriate correction procedure both in the UV-Vis and FTIR experiments. Three intermediates have been trapped at -25, -45 and -120 degrees C, which show the characteristic vibrational band pattern of the plant phytochrome phyA intermediates meta-Rc, meta-Ra and lumi-R, respectively. In the backward reaction, two intermediates corresponding to meta-F and lumi-F were trapped at -70 and -140 degrees C, respectively. FTIR spectra of all intermediates, as well as of the Pfr state, show remarkable similarities with the corresponding spectra of Cph1 phytochrome from cyanobacterium Synechocystis and the 59 kDa N-terminal fragment of Cph1, and, albeit not so pronounced, also with plant phyA. The spectral similarities and differences between the various phytochromes are discussed in terms of structural changes of the chromophore and the chromophore-protein interactions.

Functional role of the "ionic lock"--an interhelical hydrogen-bond network in family A heptahelical receptors.

Activation of family A G-protein-coupled receptors involves a rearrangement of a conserved interhelical cytoplasmic hydrogen bond network between the E(D)RY motif on transmembrane helix 3 (H3) and residues on H6, which is commonly termed the cytoplasmic "ionic lock." Glu134(3.49) of the E(D)RY motif also forms an intrahelical salt bridge with neighboring Arg135(3.50) in the dark-state crystal structure of rhodopsin. We examined the roles of Glu134(3.49) and Arg135(3.50) on H3 and Glu247(6.30) and Glu249(6.32) on H6 on the activation of rhodopsin using Fourier transform infrared spectroscopy of wild-type and mutant pigments reconstituted into lipid membranes. Activation of rhodopsin is pH-dependent with proton uptake during the transition from the inactive Meta I to the active Meta II state. Glu134(3.49) of the ERY motif is identified as the proton-accepting group, using the Fourier transform infrared protonation signature and the absence of a pH dependence of activation in the E134Q mutant. Neutralization of Arg135(3.50) similarly leads to pH-independent receptor activation, but with structural alterations in the Meta II state. Neutralization of Glu247(6.30) and Glu249(6.32) on H6, which are involved in interhelical interactions with H3 and H7, respectively, led to a shift toward Meta II in the E247Q and E249Q mutants while retaining the pH sensitivity of the equilibrium. Disruption of the interhelical interaction of Glu247(6.30) and Glu249(6.32) on H6 with H3 and H7 plays its role during receptor activation, but neutralization of the intrahelical salt bridge between Glu134(3.49) and Arg135(3.50) is considerably more critical for shifting the photoproduct equilibrium to the active conformation. These conclusions are discussed in the context of recent structural data of the beta(2)-adrenergic receptor.

FTIR study of the photoinduced processes of plant phytochrome phyA using isotope-labeled bilins and density functional theory calculations.

Fourier transform infrared spectroscopy was used to analyze the chromophore structure in the parent states Pr and Pfr of plant phytochrome phyA and the respective photoproducts lumi-R and lumi-F. The spectra were obtained from phyA adducts assembled with either uniformly or selectively isotope-labeled phytochromobilin and phycocyanobilin. The interpretation of the experimental spectra is based on the spectra of chromophore models calculated by density functional theory. Global (13)C-labeling of the tetrapyrrole allows for the discrimination between chromophore and protein bands in the Fourier transform infrared difference spectra. All infrared difference spectra display a prominent difference band attributable to a stretching mode with large contributions from the methine bridge between the inner pyrrole rings (B-C stretching). Due to mode coupling, frequencies and isotopic shifts of this mode suggest that the Pr chromophore may adopt a distorted ZZZssa or ZZZasa geometry with a twisted A-B methine bridge. The transition to lumi-R is associated with only minor changes of the amide I bands indicating limited protein structural changes during the isomerization site of the C-D methine bridge. Major protein structural changes occur upon the transition to Pfr in which the chromophore adopts a ZZEssa or ZZEasa-like state. In addition, specific interactions with the protein alter the structure of the B-C methine bridge as concluded from the substantial downshift of the respective stretching mode. These interactions are removed during the photoreaction to lumi-F (ZZE-->ZZZ), which involves only small protein structural changes.

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.

The chromophore structures of the Pr States in plant and bacterial phytochromes.

The resonance Raman spectra of the Pr state of the N-terminal 65-kDa fragment of plant phytochrome phyA have been measured and analyzed in terms of the configuration and conformation of the tetrapyrroles methine bridges. Spectra were obtained from phyA adducts reconstituted with the natural chromophore phytochromobilin as well as phycocyanobilin and its isotopomers labeled at the terminal methine bridges through (13)C/(12)C and D/H substitution. Upon comparing the resonance Raman spectra of the various phyA adducts, it was possible to identify the bands that originate from normal modes dominated by the stretching coordinates of the terminal methine bridges A-B and C-D. Quantum chemical calculations of the isolated tetrapyrroles reveal that these modes are sensitive indicators for the methine bridge configuration and conformation. For all phyA adducts, the experimental spectra of Pr including this marker band region are well reproduced by the calculated spectra obtained for the ZZZasa configuration. In contrast, there are substantial discrepancies between the experimental spectra and the spectra calculated for the ZZZssa configuration, which has been previously shown to be the chromophore geometry in the Pr state of the bacterial, biliverdin-binding phytochrome from Deinococcus radiodurans (Wagner, J. R., J. S. Brunzelle, K. T. Forest, R. D. Vierstra. 2005. Nature. 438:325-331). The results of this work, therefore, suggest that plant and bacterial (biliverdin-binding) phytochromes exhibit different structures in the parent state although the mechanism of the photoinduced reaction cycle may be quite similar.

Interaction of a G protein-coupled receptor with a G protein-derived peptide induces structural changes in both peptide and receptor: a Fourier-transform infrared study using isotopically labeled peptides.

G protein-coupled receptor signaling involves productive interaction between agonist-activated receptor and G protein. We have used Fourier-transform infrared difference spectroscopy to examine the interaction between the active Meta II state of the visual pigment rhodopsin with a peptide analogue corresponding to the C terminus of the alpha-subunit of the G protein transducin. Formation of the receptor-peptide complex evokes a spectral signature consisting of conformationally sensitive amide I and amide II difference bands. In order to distinguish between amide backbone contributions of the peptide and of the receptor moiety to the vibrational spectra, we employed complete (13)C,(15)N-labeling of the peptide. This isotopic labeling downshifts selectively the bands of the peptide, which can thus be extracted. Our results show that formation of the complex between the activated Meta II receptor state and the peptide is accompanied by structural changes of the peptide, and of the receptor, indicating that the conformation of the Meta II.peptide complex is different from that of Meta II. This result implies that the activated receptor state has conformational flexibility. Binding of the peptide to the activated receptor state stabilizes a substate that deviates from that stabilized only by the agonist.

Anion uptake in halorhodopsin from Natromonas pharaonis studied by FTIR spectroscopy: consequences for the anion transport mechanism.

The uptake of chloride, bromide, iodide, nitrate, and azide by anion-depleted blue halorhodopsin from Natronobacterium pharaonis has been followed by FTIR difference spectroscopy using an ATR sampling device. The spectra are compared with the spectrum of the O intermediate obtained by time-resolved FTIR studies of the photocycle. It is demonstrated that anion-free blue halorhodopsin can be identified with the O intermediate and, thus, that the decay of O is due to the passive uptake of the anion. The great similarity of the anion-binding spectra and their identity in the case of the monoatomic anions indicate a rather unspecific binding site for the different anions dominated by electrostatic interactions. Comparing spectra obtained with 15N nitrate and unlabeled nitrate, the NO-stretching bands could be identified. The small splitting and the small IR intensity of those bands indicate a rather nonpolar binding site with a rather isotropic influence on the nitrate, in contrast to aqueous nitrate. In further experiments on the photocycle of blue halorhodopsin, the all-trans --> 13-cis isomerization can be clearly identified. Up to 100 micros, the isomerization-induced structural changes deduced from amide I changes are similar to those occurring during the anion-transporting photocycle. Compared to these, the molecular changes involved in the release and their reversion during the uptake of anions are considerably larger. They can be reached via two pathways: (1) by reducing the anion concentration and (2) transiently during the anion-transporting photocycle with the formation of the precursor of O with O conformation. Consequences of the anion transport mechanism are discussed.

Modulating rhodopsin receptor activation by altering the pKa of the retinal Schiff base.

The visual pigment rhodopsin is a seven-transmembrane (7-TM) G protein-coupled receptor (GPCR). Activation of rhodopsin involves two pH-dependent steps: proton uptake at a conserved cytoplasmic motif between TM helices 3 and 6, and disruption of a salt bridge between a protonated Schiff base (PSB) and its carboxylate counterion in the transmembrane core of the receptor. Formation of an artificial pigment with a retinal chromophore fluorinated at C14 decreases the intrinsic pKa of the PSB and thereby destabilizes this salt bridge. Using Fourier transform infrared difference and UV-visible spectroscopy, we characterized the pH-dependent equilibrium between the active photoproduct Meta II and its inactive precursor, Meta I, in the 14-fluoro (14-F) analogue pigment. The 14-F chromophore decreases the enthalpy change of the Meta I-to-Meta II transition and shifts the Meta I/Meta II equilibrium toward Meta II. Combining C14 fluorination with deletion of the retinal beta-ionone ring to form a 14-F acyclic artificial pigment uncouples disruption of the Schiff base salt bridge from transition to Meta II and in particular from the cytoplasmic proton uptake reaction, as confirmed by combining the 14-F acyclic chromophore with the E134Q mutant. The 14-F acyclic analogue formed a stable Meta I state with a deprotonated Schiff base and an at least partially protonated protein counterion. The combination of retinal modification and site-directed mutagenesis reveals that disruption of the protonated Schiff base salt bridge is the most important step thermodynamically in the transition from Meta I to Meta II. This finding is particularly important since deprotonation of the retinal PSB is known to precede the transition to the active state in rhodopsin activation and is consistent with models of agonist-dependent activation of other GPCRs.

Time-resolved rapid-scan Fourier transform infrared difference spectroscopy on a noncyclic photosystem: rhodopsin photointermediates from Lumi to Meta II.

The visual pigment rhodopsin has been extensively studied for the kinetics of its photointermediates by various spectroscopic methods. Unlike such archaeal retinal proteins as bacteriorhodopsin, visual rhodopsin does not thermally recover its dark state after photoexcitation, which precludes repeated excitation of a single sample and thereby complicates time-resolved experiments. Kinetic data on the late rhodopsin photointermediates have so far been available mainly from time-resolved ultraviolet (UV)-visible spectroscopy, but not from Fourier transform infrared (FTIR) spectroscopy. The latter has the advantage of being informative of structural changes of both chromophore and protein, but does not allow the highly reproducible, automated sample exchange procedures available to UV-visible spectroscopy. Using rapid-scan FTIR difference spectroscopy, we obtained time-resolved data sets that were analyzed by a maximum entropy inverse Laplace-transform. Covering the time range from 8 ms to 15 s at temperatures of 0 and -7 degrees C, the transitions from the Lumi to the Meta I and from the Meta I to the Meta II photoproduct states could be resolved. In the transition from Meta I to Meta II, our data reveal a partial deprotonation of the retinal Schiff base preceding the conformational change of the receptor protein to Meta II. The technique and the results are discussed in regard to its advantages as well as its limitations.