Ligand efficacy modulates conformational dynamics of the µ-opioid receptor.

The µ-opioid receptor (µOR) is an important target for pain management1 and molecular understanding of drug action on µOR will facilitate the development of better therapeutics. Here we show, using double electron-electron resonance and single-molecule fluorescence resonance energy transfer, how ligand-specific conformational changes of µOR translate into a broad range of intrinsic efficacies at the transducer level. We identify several conformations of the cytoplasmic face of the receptor that interconvert on different timescales, including a pre-activated conformation that is capable of G-protein binding, and a fully activated conformation that markedly reduces GDP affinity within the ternary complex. Interaction of ß-arrestin-1 with the µOR core binding site appears less specific and occurs with much lower affinity than binding of Gi.

A Highly Ordered Nitroxide Side Chain for Distance Mapping and Monitoring Slow Structural Fluctuations in Proteins.

Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) is an established tool for exploring protein structure and dynamics. Although nitroxide side chains attached to a single cysteine via a disulfide linkage are commonly employed in SDSL-EPR, their internal flexibility complicates applications to monitor slow internal motions in proteins and to structure determination by distance mapping. Moreover, the labile disulfide linkage prohibits the use of reducing agents often needed for protein stability. To enable the application of SDSL-EPR to the measurement of slow internal dynamics, new spin labels with hindered internal motion are desired. Here, we introduce a highly ordered nitroxide side chain, designated R9, attached at a single cysteine residue via a non-reducible thioether linkage. The reaction to introduce R9 is highly selective for solvent-exposed cysteine residues. Structures of R9 at two helical sites in T4 Lysozyme were determined by X-ray crystallography and the mobility in helical sequences was characterized by EPR spectral lineshape analysis, Saturation Transfer EPR, and Saturation Recovery EPR. In addition, interspin distance measurements between pairs of R9 residues are reported. Collectively, all data indicate that R9 will be useful for monitoring slow internal structural fluctuations, and applications to distance mapping via dipolar spectroscopy and relaxation enhancement methods are anticipated. Supplementary Information: The online version contains supplementary material available at 10.1007/s00723-023-01618-8.

Membrane potential accelerates sugar uptake by stabilizing the outward facing conformation of the Na/glucose symporter vSGLT.

Sodium-dependent glucose transporters (SGLTs) couple a downhill Na+ ion gradient to actively transport sugars. Here, we investigate the impact of the membrane potential on vSGLT structure and function using sugar uptake assays, double electron-electron resonance (DEER), electrostatic calculations, and kinetic modeling. Negative membrane potentials, as present in all cell types, shift the conformational equilibrium of vSGLT towards an outward-facing conformation, leading to increased sugar transport rates. Electrostatic calculations identify gating charge residues responsible for this conformational shift that when mutated reduce galactose transport and eliminate the response of vSGLT to potential. Based on these findings, we propose a comprehensive framework for sugar transport via vSGLT, where the cellular membrane potential facilitates resetting of the transporter after cargo release. This framework holds significance not only for SGLTs but also for other transporters and channels.

Dynamics of the Second Extracellular Loop Control Transducer Coupling of Peptide-Activated GPCRs.

Many peptide-activated rhodopsin-like GPCRs share a ß-hairpin folding motif in the extracellular loop 2 (ECL2), which interacts with the peptide ligand while at the same time being connected to transmembrane helix 3 (TM3) via a highly conserved disulfide bond. Currently, it remains unknown whether the coupling of the specifically shaped ECL2 to TM3 influences the activation of peptide-activated GPCRs. We investigated this possibility in a selection of peptide GPCRs with known structures. Most of the receptors with cysteine to alanine mutations folded like the respective wild-type and resided in the cell membrane, challenging pure folding stabilization by the disulfide bridge. G-protein signaling of the disulfide mutants was retained to a greater extent in secretin-like GPCRs than in rhodopsin-like GPCRs, while recruitment of arrestin was completely abolished in both groups, which may be linked to alterations in ligand residence time. We found a correlation between receptor activity of the neuropeptide Y2 receptor and alterations in ECL2 dynamics using engineered disulfide bridges or site-directed spin labeling and EPR spectroscopy. These data highlight the functional importance of the TM3-ECL2 link for the activation of specific signaling pathways in peptide-activated GPCRs, which might have implications for future drug discovery.

Conformational dynamics of the µ-opioid receptor determine ligand intrinsic efficacy.

The µ-opioid receptor (µOR) is an important target for pain management and the molecular understanding of drug action will facilitate the development of better therapeutics. Here we show, using double electron-electron resonance (DEER) and single-molecule fluorescence resonance energy transfer (smFRET), how ligand-specific conformational changes of the µOR translate into a broad range of intrinsic efficacies at the transducer level. We identify several cytoplasmic receptor conformations interconverting on different timescales, including a pre-activated receptor conformation which is capable of G protein binding, and a fully activated conformation which dramatically lowers GDP affinity within the ternary complex. Interaction of ß-arrestin-1 with the µOR core binding site appears less specific and occurs with much lower affinity than binding of G protein Gi.

DEER Analysis of GPCR Conformational Heterogeneity.

G protein-coupled receptors (GPCRs) represent a large class of transmembrane helical proteins which are involved in numerous physiological signaling pathways and therefore represent crucial pharmacological targets. GPCR function and the action of therapeutic molecules are defined by only a few parameters, including receptor basal activity, ligand affinity, intrinsic efficacy and signal bias. These parameters are encoded in characteristic receptor conformations existing in equilibrium and their populations, which are thus of paramount interest for the understanding of receptor (mal-)functions and rational design of improved therapeutics. To this end, the combination of site-directed spin labeling and EPR spectroscopy, in particular double electron-electron resonance (DEER), is exceedingly valuable as it has access to sub-Angstrom spatial resolution and provides a detailed picture of the number and populations of conformations in equilibrium. This review gives an overview of existing DEER studies on GPCRs with a focus on the delineation of structure/function frameworks, highlighting recent developments in data analysis and visualization. We introduce "conformational efficacy" as a parameter to describe ligand-specific shifts in the conformational equilibrium, taking into account the loose coupling between receptor segments observed for different GPCRs using DEER.

Viewing rare conformations of the ß2 adrenergic receptor with pressure-resolved DEER spectroscopy.

The ß2 adrenergic receptor (ß2AR) is an archetypal G protein coupled receptor (GPCR). One structural signature of GPCR activation is a large-scale movement (ca. 6 to 14 Å) of transmembrane helix 6 (TM6) to a conformation which binds and activates a cognate G protein. The ß2AR exhibits a low level of agonist-independent G protein activation. The structural origin of this basal activity and its suppression by inverse agonists is unknown but could involve a unique receptor conformation that promotes G protein activation. Alternatively, a conformational selection model proposes that a minor population of the canonical active receptor conformation exists in equilibrium with inactive forms, thus giving rise to basal activity of the ligand-free receptor. Previous spin-labeling and fluorescence resonance energy transfer experiments designed to monitor the positional distribution of TM6 did not detect the presence of the active conformation of ligand-free ß2AR. Here we employ spin-labeling and pressure-resolved double electron-electron resonance spectroscopy to reveal the presence of a minor population of unliganded receptor, with the signature outward TM6 displacement, in equilibrium with inactive conformations. Binding of inverse agonists suppresses this population. These results provide direct structural evidence in favor of a conformational selection model for basal activity in ß2AR and provide a mechanism for inverse agonism. In addition, they emphasize 1) the importance of minor populations in GPCR catalytic function; 2) the use of spin-labeling and variable-pressure electron paramagnetic resonance to reveal them in a membrane protein; and 3) the quantitative evaluation of their thermodynamic properties relative to the inactive forms, including free energy, partial molar volume, and compressibility.

Synthetic nanobodies as angiotensin receptor blockers.

There is considerable interest in developing antibodies as functional modulators of G protein-coupled receptor (GPCR) signaling for both therapeutic and research applications. However, there are few antibody ligands targeting GPCRs outside of the chemokine receptor group. GPCRs are challenging targets for conventional antibody discovery methods, as many are highly conserved across species, are biochemically unstable upon purification, and possess deeply buried ligand-binding sites. Here, we describe a selection methodology to enrich for functionally modulatory antibodies using a yeast-displayed library of synthetic camelid antibody fragments called "nanobodies." Using this platform, we discovered multiple nanobodies that act as antagonists of the angiotensin II type 1 receptor (AT1R). Following angiotensin II infusion in mice, we found that an affinity matured nanobody antagonist has comparable antihypertensive activity to the angiotensin receptor blocker (ARB) losartan. The unique pharmacology and restricted biodistribution of nanobody antagonists may provide a path for treating hypertensive disorders when small-molecule drugs targeting the AT1R are contraindicated, for example, in pregnancy.

Evaluation of left ventricular function in patients with acute ischaemic stroke using cine cardiovascular magnetic resonance imaging.

AIMS: Heart failure (HF) is frequent in patients with acute ischaemic stroke (AIS) and associated with higher morbidity and mortality. Assessment of cardiac function in AIS patients using cardiovascular MRI (CMR) may help to detect HF. We report the rate of systolic and diastolic dysfunction in a cohort of patients with AIS using CMR and compare cine real-time (CRT) sequences with the reference of segmented cine steady-state free precession sequences. METHODS AND RESULTS: Patients with AIS without known atrial fibrillation were prospectively enrolled in the HEart and BRain Interfaces in Acute Ischemic Stroke (HEBRAS) study (NCT02142413) and underwent CMR at 3 Tesla within 7 days after AIS. Validity of CRT sequences was determined in 50 patients. A total of 229 patients were included in the analysis (mean age 66 years; 35% women; HF 2%). Evaluation of cardiac function was successful in 172 (75%) patients. Median time from stroke onset to CMR was 82 h (interquartile range 56-111) and 54 h (interquartile range 31-78) from cerebral MRI to CMR. Systolic dysfunction was observed in 43 (25%) and diastolic dysfunction in 102 (59%) patients. Diagnostic yield was similar using CRT or segmented cine imaging (no significant difference in left ventricular ejection fraction, myocardial mass, time to peak filling rate, and peak filling rate ratio E/A). Intraobserver and interobserver agreement was high (κ = 0.78-1.0 for all modalities). CONCLUSIONS: Cardiovascular MRI at 3 Tesla is an appropriate method for the evaluation of cardiac function in a selected cohort of patients with AIS. Systolic and diastolic dysfunction is frequent in these patients. CRT imaging allows reliable assessment of systolic and diastolic function.

Diagnosis of Left Ventricular Diastolic Dysfunction Using Cardiac Magnetic Resonance Imaging: Comparison of Volume-Time Curves Derived from Long- and Short-Axis Cine Steady-State Free Precession Datasets.

PURPOSE: To evaluate the diagnostic performance of diastolic function parameters derived from long-axis (LAX) planimetry compared with short-axis (SAX) volumetry in cardiac magnetic resonance imaging. MATERIALS AND METHODS: Cine steady-state free precession (SSFP) datasets of 15 healthy participants (8 young and 7 middle aged) and 25 patients with echocardiographically proven diastolic dysfunction (9 mild, 9 moderate, and 7 severe) were retrospectively included. Volume-time curves for assessing left ventricular (LV) function were obtained by manually contouring the LV endocardial borders in SAX and LAX datasets. The time needed for contouring was recorded for each dataset. The following LV parameters were determined: end-diastolic volume (EDV), end-systolic volume (ESV), ejection fraction (EF), myocardial mass (MM), time to peak filling rate (TPFR), normalized peak filling rate (nPFR), and the ratio of early to late peak filling rate (E/A ratio). A Wilcoxon signed-rank test was used to compare subgroups based on age and severity of diastolic dysfunction for statistical differences. Intraclass correlation coefficients were used to assess intermethod and interobserver reliability. RESULTS: Accuracy for the diagnosis of diastolic dysfunction was highest for E/A (mild diastolic dysfunction) and nPFR (any stage of diastolic dysfunction) derived from LAX datasets (E/A: area under the curve (AUC) = 0.97, sensitivity of 68 % and specificity of 100 %; nPFR: AUC = 0.84, sensitivity of 84 % and specificity of 80 %). Diastolic parameters showed a moderate to good intraclass correlation between both methods. The mean differences in EDV, ESV, EF, and MM were 5.3 ml, 1.9 ml, 3.5 %, and 11 g, respectively (each p < 0.001). Significantly less time was needed to derive volume-time curves from LAX images (median 14:45 min, interquartile range 14:15-15:53 min versus median 29:25 min, interquartile range 28:12-32:22 min; p = 0.001). The interobserver reliability was generally good to excellent. CONCLUSION: Diastolic function parameters derived from left ventricular LAX planimetry have high diagnostic performance and can be obtained in significantly less time compared with SAX volumetry. These findings may pave the way for routine use of LAX planimetry in the clinical diagnosis of diastolic dysfunction. KEY POINTS: · Diastolic function parameters derived from long-axis datasets have high diagnostic performance.. · Generation of volume-time curves using long-axis datasets requires significantly less time.. · This time savings may allow use of cardiac MRI for the diagnosis of diastolic dysfunction in the clinical routine.. CITATION FORMAT: · Schaafs LA, Wyschkon S, Elgeti M et al. Diagnosis of Left Ventricular Diastolic Dysfunction Using Cardiac Magnetic Resonance Imaging: Comparison of Volume-Time Curves Derived from Long- and Short-Axis Cine Steady-State Free Precession Datasets. Fortschr Röntgenstr 2020; 192: 764 - 775.

Angiotensin Analogs with Divergent Bias Stabilize Distinct Receptor Conformations.

"Biased" G protein-coupled receptor (GPCR) agonists preferentially activate pathways mediated by G proteins or ß-arrestins. Here, we use double electron-electron resonance spectroscopy to probe the changes that ligands induce in the conformational distribution of the angiotensin II type I receptor. Monitoring distances between 10 pairs of nitroxide labels distributed across the intracellular regions enabled mapping of four underlying sets of conformations. Ligands from different functional classes have distinct, characteristic effects on the conformational heterogeneity of the receptor. Compared to angiotensin II, the endogenous agonist, agonists with enhanced Gq coupling more strongly stabilize an "open" conformation with an accessible transducer-binding site. ß-arrestin-biased agonists deficient in Gq coupling do not stabilize this open conformation but instead favor two more occluded conformations. These data suggest a structural mechanism for biased ligand action at the angiotensin receptor that can be exploited to rationally design GPCR-targeting drugs with greater specificity of action.

The arrestin-1 finger loop interacts with two distinct conformations of active rhodopsin.

Signaling of the prototypical G protein-coupled receptor (GPCR) rhodopsin through its cognate G protein transducin (Gt) is quenched when arrestin binds to the activated receptor. Although the overall architecture of the rhodopsin/arrestin complex is known, many questions regarding its specificity remain unresolved. Here, using FTIR difference spectroscopy and a dual pH/peptide titration assay, we show that rhodopsin maintains certain flexibility upon binding the "finger loop" of visual arrestin (prepared as synthetic peptide ArrFL-1). We found that two distinct complexes can be stabilized depending on the protonation state of E3.49 in the conserved (D)ERY motif. Both complexes exhibit different interaction modes and affinities of ArrFL-1 binding. The plasticity of the receptor within the rhodopsin/ArrFL-1 complex stands in contrast to the complex with the C terminus of the Gt α-subunit (GαCT), which stabilizes only one specific substate out of the conformational ensemble. However, Gt α-subunit binding and both ArrFL-1-binding modes involve a direct interaction to conserved R3.50, as determined by site-directed mutagenesis. Our findings highlight the importance of receptor conformational flexibility and cytoplasmic proton uptake for modulation of rhodopsin signaling and thereby extend the picture provided by crystal structures of the rhodopsin/arrestin and rhodopsin/ArrFL-1 complexes. Furthermore, the two binding modes of ArrFL-1 identified here involve motifs of conserved amino acids, which indicates that our results may have elucidated a common modulation mechanism of class A GPCR-G protein/-arrestin signaling.

The Activation Pathway of Human Rhodopsin in Comparison to Bovine Rhodopsin.

Rhodopsin, the photoreceptor of rod cells, absorbs light to mediate the first step of vision by activating the G protein transducin (Gt). Several human diseases, such as retinitis pigmentosa or congenital night blindness, are linked to rhodopsin malfunctions. Most of the corresponding in vivo studies and structure-function analyses (e.g. based on protein x-ray crystallography or spectroscopy) have been carried out on murine or bovine rhodopsin. Because these rhodopsins differ at several amino acid positions from human rhodopsin, we conducted a comprehensive spectroscopic characterization of human rhodopsin in combination with molecular dynamics simulations. We show by FTIR and UV-visible difference spectroscopy that the light-induced transformations of the early photointermediates are very similar. Significant differences between the pigments appear with formation of the still inactive Meta I state and the transition to active Meta II. However, the conformation of Meta II and its activity toward the G protein are essentially the same, presumably reflecting the evolutionary pressure under which the active state has developed. Altogether, our results show that although the basic activation pathways of human and bovine rhodopsin are similar, structural deviations exist in the inactive conformation and during receptor activation, even between closely related rhodopsins. These differences between the well studied bovine or murine rhodopsins and human rhodopsin have to be taken into account when the influence of point mutations on the activation pathway of human rhodopsin are investigated using the bovine or murine rhodopsin template sequences.

Structure-based biophysical analysis of the interaction of rhodopsin with G protein and arrestin.

In this chapter, we describe a set of complementary techniques that we use to study the activation of rhodopsin, a G protein-coupled receptor (GPCR), and its functional interactions with G protein and arrestin. The protein reagents used for these studies come from native disc membranes or heterologous expression, and G protein and arrestin are often replaced with less complex synthetic peptides derived from key interaction sites of these binding partners (BPs). We first report on our approach to protein X-ray crystallography and describe how protein crystals from native membranes are obtained. The crystal structures provide invaluable resolution, but other techniques are required to assess the dynamic equilibria characteristic for active GPCRs. The simplest approach is "Extra Meta II," which uses UV/Vis absorption spectroscopy to monitor the equilibrium of photoactivated states. Site-specific information about the BPs (e.g., arrestin) is added by fluorescence techniques employing mutants labeled with reporter groups. All functional changes in both the receptor and interacting proteins or peptides are seen with highest precision using Fourier transform infrared (FTIR) difference spectroscopy. In our approach, the lack of site-specific information in FTIR is overcome by parallel molecular dynamics simulations, which are employed to interpret the results and to extend the timescale down to the range of conformational substates.

Crystal structure of a common GPCR-binding interface for G protein and arrestin.

G-protein-coupled receptors (GPCRs) transmit extracellular signals to activate intracellular heterotrimeric G proteins (Gαßγ) and arrestins. For G protein signalling, the Gα C-terminus (GαCT) binds to a cytoplasmic crevice of the receptor that opens upon activation. A consensus motif is shared among GαCT from the Gi/Gt family and the 'finger loop' region (ArrFL1-4) of all four arrestins. Here we present a 2.75 Å crystal structure of ArrFL-1, a peptide analogue of the finger loop of rod photoreceptor arrestin, in complex with the prototypical GPCR rhodopsin. Functional binding of ArrFL to the receptor was confirmed by ultraviolet-visible absorption spectroscopy, competitive binding assays and Fourier transform infrared spectroscopy. For both GαCT and ArrFL, binding to the receptor crevice induces a similar reverse turn structure, although significant structural differences are seen at the rim of the binding crevice. Our results reflect both the common receptor-binding interface and the divergent biological functions of G proteins and arrestins.

Position of transmembrane helix 6 determines receptor G protein coupling specificity.

G protein coupled receptors (GPCRs) transmit extracellular signals into the cell by binding and activating different intracellular signaling proteins, such as G proteins (Gαßγ, families Gi, Gs, Gq, G12/13) or arrestins. To address the issue of Gs vs Gi coupling specificity, we carried out molecular dynamics simulations of lipid-embedded active ß2-adrenoceptor (ß2AR*) in complex with C-terminal peptides derived from the key interaction site of Gα (GαCT) as surrogate of Gαßγ. We find that GiαCT and GsαCT exploit distinct cytoplasmic receptor conformations that coexist in the uncomplexed ß2AR*. The slim GiαCT stabilizes a ß2AR* conformation, not accessible to the bulkier GsαCT, which requires a larger TM6 outward tilt for binding. Our results suggest that the TM6 conformational heterogeneity regulates the catalytic activity of ß2AR* toward Gi or Gs.

Precision vs flexibility in GPCR signaling.

The G protein coupled receptor (GPCR) rhodopsin activates the heterotrimeric G protein transducin (Gt) to transmit the light signal into retinal rod cells. The rhodopsin activity is virtually zero in the dark and jumps by more than one billion fold after photon capture. Such perfect switching implies both high fidelity and speed of rhodopsin/Gt coupling. We employed Fourier transform infrared (FTIR) spectroscopy and supporting all-atom molecular dynamics (MD) simulations to study the conformational diversity of rhodopsin in membrane environment and extend the static picture provided by the available crystal structures. The FTIR results show how the equilibria of inactive and active protein states of the receptor (so-called metarhodopsin states) are regulated by the highly conserved E(D)RY and Yx7K(R) motives. The MD data identify an intrinsically unstructured cytoplasmic loop region connecting transmembrane helices 5 and 6 (CL3) and show how each protein state is split into conformational substates. The C-termini of the Gtγ- and Gtα-subunits (GαCT and GγCT), prepared as synthetic peptides, are likely to bind sequentially and at different sites of the active receptor. The peptides have different effects on the receptor conformation. While GγCT stabilizes the active states but preserves CL3 flexibility, GαCT selectively stabilizes a single conformational substate with largely helical CL3, as it is found in crystal structures. Based on these results we propose a mechanism for the fast and precise signal transfer from rhodopsin to Gt, which assumes a stepwise and mutual reduction of their conformational space. The mechanism relies on conserved amino acids and may therefore underlie GPCR/G protein coupling in general.

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.

Activity switches of rhodopsin.

Rhodopsin, the visual pigment of the rod photoreceptor cell contains as its light-sensitive cofactor 11-cis retinal, which is bound by a protonated Schiff base between its aldehyde group and the Lys296 side chain of the apoprotein. Light activation is achieved by 11-cis to all-trans isomerization and subsequent thermal relaxation into the active, G protein-binding metarhodopsin II state. Metarhodopsin II decays via two parallel pathways, which both involve hydrolysis of the Schiff base eventually to opsin and released all-trans retinal. Subsequently, rhodopsin's dark state is regenerated by a complicated retinal metabolism, termed the retinoid cycle. Unlike other retinal proteins, such as bacteriorhodopsin, this regeneration cycle cannot be short cut by light, because blue illumination of active metarhodopsin II does not lead back to the ground state but to the formation of largely inactive metarhodopsin III. In this review, mechanistic details of activating and deactivating pathways of rhodopsin, particularly concerning the roles of the retinal, are compared. Based on static and time-resolved UV/Vis and FTIR spectroscopic data, we discuss a model of the light-induced deactivation. We describe properties and photoreactions of metarhodopsin III and suggest potential roles of this intermediate for vision.

Deactivation and proton transfer in light-induced metarhodopsin II/metarhodopsin III conversion: a time-resolved fourier transform infrared spectroscopic study.

Vertebrate rhodopsin shares with other retinal proteins the 11-cis-retinal chromophore and the light-induced 11-cis/trans isomerization triggering its activation pathway. However, only in rhodopsin the retinylidene Schiff base bond to the apoprotein is eventually hydrolyzed, making a complex regeneration pathway necessary. Metabolic regeneration cannot be short-cut, and light absorption in the active metarhodopsin (Meta) II intermediate causes anti/syn isomerization around the retinylidene linkage rather than reversed trans/cis isomerization. A new deactivating pathway is thereby triggered, which ends in the Meta III "retinal storage" product. Using time-resolved Fourier transform infrared spectroscopy, we show that the identified steps of receptor activation, including Schiff base deprotonation, protein structural changes, and proton uptake by the apoprotein, are all reversed. However, Schiff base reprotonation is much faster than the activating deprotonation, whereas the protein structural changes are slower. The final proton release occurs with pK approximately 4.5, similar to the pK of a free Glu residue and to the pK at which the isolated opsin apoprotein becomes active. A forced deprotonation, equivalent to the forced protonation in the activating pathway, which occurs against the unfavorable pH of the medium, is not observed. This explains properties of the final Meta III product, which displays much higher residual activity and is less stable than rhodopsin arising from regeneration with 11-cis-retinal. We propose that the anti/syn conversion can only induce a fast reorientation and distance change of the Schiff base but fails to build up the full set of dark ground state constraints, presumably involving the Glu(134)/Arg(135) cluster.