Insight into the chromophore of rhodopsin and its Meta-II photointermediate by 19F solid-state NMR and chemical shift tensor calculations.

19F nuclei are useful labels in solid-state NMR studies, since their chemical shift and tensor elements are very sensitive to the electrostatic and space-filling properties of their local environment. In this study we have exploited a fluorine substituent, strategically placed at the C-12-position of 11-cis retinal, the chromophore of visual rhodopsins. This label was used to explore the local environment of the chromophore in the ground state of bovine rhodopsin and its active photo-intermediate Meta II. In addition, the chemical shift and tensor elements of the chromophore in the free state in a membrane environment and the bound state in the protein were determined. Upon binding of the chromophore into rhodopsin and Meta II, the isotropic chemical shift changes in the opposite direction by +9.7 and -8.4 ppm, respectively. An unusually large isotropic shift difference of 35.9 ppm was observed between rhodopsin and Meta II. This partly originates in the light-triggered 11-cis to all-trans isomerization of the chromophore. The other part reflects the local conformational rearrangements in the chromophore and the binding pocket. These NMR data were correlated with the available X-ray structures of rhodopsin and Meta II using bond polarization theory. For this purpose hydrogen atoms have to be inserted and hereto a family of structures were derived that best correlated with the well-established 13C chemical shifts. Based upon these structures, a 12-F derivative was obtained that best corresponded with the experimentally determined 19F chemical shifts and tensor elements. The combined data indicate strong changes in the local environment of the C-12 position and a substantially different interaction pattern with the protein in Meta II as compared to rhodopsin.

Structural Changes in an Anion Channelrhodopsin: Formation of the K and L Intermediates at 80 K.

A recently discovered natural family of light-gated anion channelrhodopsins (ACRs) from cryptophyte algae provides an effective means of optogenetically silencing neurons. The most extensively studied ACR is from Guillardia theta (GtACR1). Earlier studies of GtACR1 have established a correlation between formation of a blue-shifted L-like intermediate and the anion channel "open" state. To study structural changes of GtACR1 in the K and L intermediates of the photocycle, a combination of low-temperature Fourier transform infrared (FTIR) and ultraviolet-visible absorption difference spectroscopy was used along with stable-isotope retinal labeling and site-directed mutagenesis. In contrast to bacteriorhodopsin (BR) and other microbial rhodopsins, which form only a stable red-shifted K intermediate at 80 K, GtACR1 forms both stable K and L-like intermediates. Evidence includes the appearance of positive ethylenic and fingerprint vibrational bands characteristic of the L intermediate as well as a positive visible absorption band near 485 nm. FTIR difference bands in the carboxylic acid C═O stretching region indicate that several Asp/Glu residues undergo hydrogen bonding changes at 80 K. The Glu68 → Gln and Ser97 → Glu substitutions, residues located close to the retinylidene Schiff base, altered the K:L ratio and several of the FTIR bands in the carboxylic acid region. In the case of the Ser97 → Glu substitution, a significant red-shift of the absorption wavelength of the K and L intermediates occurs. Sequence comparisons suggest that L formation in GtACR1 at 80 K is due in part to the substitution of the highly conserved Leu or Ile at position 93 in helix 3 (BR sequence) with the homologous Met105 in GtACR1.

Coupled HOOP signature correlates with quantum yield of isorhodopsin and analog pigments.

With a quantum yield of 0.66±0.03 the photoisomerization efficiency of the visual pigment rhodopsin (11-cis⇒all-trans chromophore) is exceptionally high. This is currently explained by coherent coupling of the excited state electronic wavepacket with local vibrational nuclear modes, facilitating efficient cross-over at a conical intersection onto the photoproduct energy surface. The 9-cis counterpart of rhodopsin, dubbed isorhodopsin, has a much lower quantum yield (0.26±0.03), which, however, can be markedly enhanced by modification of the retinal chromophore (7,8-dihydro and 9-cyclopropyl derivatives). The coherent coupling in the excited state is promoted by torsional skeletal and coupled HOOP vibrational modes, in combination with a twisted conformation around the isomerization region. Since such torsion will strongly enhance the infrared intensity of coupled HOOP modes, we investigated FTIR difference spectra of rhodopsin, isorhodopsin and several analog pigments in the spectral range of isolated and coupled HCCH wags. As a result we propose that the coupled HOOP signature in these retinal pigments correlates with the distribution of torsion over counteracting segments in the retinylidene polyene chain. As such the HOOP signature can act as an indicator for the photoisomerization efficiency, and can explain the higher quantum yield of the 7,8-dihydro and 9-cyclopropyl-isorhodopsin analogs.

Modulation of spectral properties and pump activity of proteorhodopsins by retinal analogues.

Proteorhodopsins are heptahelical membrane proteins which function as light-driven proton pumps. They use all-trans-retinal A1 as a ligand and chromophore and absorb visible light (520-540 nm). In the present paper, we describe modulation of the absorbance band of the proteorhodopsin from Monterey Bay SAR 86 gammaproteobacteria (PR), its red-shifted double mutant PR-D212N/F234S (PR-DNFS) and Gloeobacter rhodopsin (GR). This was approached using three analogues of all-trans-retinal A1, which differ in their electronic and conformational properties: all-trans-6,7-s-trans-locked retinal A1, all-trans-phenyl-retinal A1 and all-trans-retinal A2. We further probed the effect of these retinal analogues on the proton pump activity of the proteorhodopsins. Our results indicate that, whereas the constraints of the retinal-binding pocket differ for the proteorhodopsins, at least two of the retinal analogues are capable of shifting the absorbance bands of the pigments either bathochromically or hypsochromically, while maintaining their proton pump activity. Furthermore, the shifts implemented by the analogues add up to the shift induced by the double mutation in PR-DNFS. This type of chromophore substitution may present attractive applications in the field of optogenetics, towards increasing the flexibility of optogenetic tools or for membrane potential probes.

Comparison of the structural changes occurring during the primary phototransition of two different channelrhodopsins from Chlamydomonas algae.

Channelrhodopsins (ChRs) from green flagellate algae function as light-gated ion channels when expressed heterologously in mammalian cells. Considerable interest has focused on understanding the molecular mechanisms of ChRs to bioengineer their properties for specific optogenetic applications such as elucidating the function of specific neurons in brain circuits. While most studies have used channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2), in this work low-temperature Fourier transform infrared-difference spectroscopy is applied to study the conformational changes occurring during the primary phototransition of the red-shifted ChR1 from Chlamydomonas augustae (CaChR1). Substitution with isotope-labeled retinals or the retinal analogue A2, site-directed mutagenesis, hydrogen-deuterium exchange, and H2(18)O exchange were used to assign bands to the retinal chromophore, protein, and internal water molecules. The primary phototransition of CaChR1 at 80 K involves, in contrast to that of CrChR2, almost exclusively an all-trans to 13-cis isomerization of the retinal chromophore, as in the primary phototransition of bacteriorhodopsin (BR). In addition, significant differences are found for structural changes of the protein and internal water(s) compared to those of CrChR2, including the response of several Asp/Glu residues to retinal isomerization. A negative amide II band is identified in the retinal ethylenic stretch region of CaChR1, which reflects along with amide I bands alterations in protein backbone structure early in the photocycle. A decrease in the hydrogen bond strength of a weakly hydrogen bonded internal water is detected in both CaChR1 and CrChR2, but the bands are much broader in CrChR2, indicating a more heterogeneous environment. Mutations involving residues Glu169 and Asp299 (homologues of the Asp85 and Asp212 Schiff base counterions, respectively, in BR) lead to the conclusion that Asp299 is protonated during P1 formation and suggest that these residues interact through a strong hydrogen bond that facilitates the transfer of a proton from Glu169.

Preparation of 2'-13C-L-histidine starting from 13C-thiocyanate: synthetic access to any site-directed stable isotope enriched L-histidine.

1-Benzyl-2-(methylthio)-imidazole-5-ketone is obtained in a few simple steps starting from thiocyanate and glycine amide (glycin). Subsequent treatment with diethyl phosphorocyanidate and functional group manipulations gives 1-benzyl-5-chloromethyl-imidazolium chloride. This compound is converted under mild O'Donnell conditions into the corresponding L-histidine derivative. After deprotection L-histidine is obtained in good yield and 99% enantiomeric excess. 2'-13C-L-Histidine has been obtained via this new scheme with high (99%) 13C incorporation starting with commercially available 13C- thiocyanate. This synthetic scheme allows access to any isotopomer of L-histidine and many other biologically important imidazole derivatives.

Quantification of globotriaosylsphingosine in plasma and urine of fabry patients by stable isotope ultraperformance liquid chromatography-tandem mass spectrometry.

BACKGROUND: Biochemical markers that accurately reflect the severity and progression of disease in patients with Fabry disease and their response to treatment are urgently needed. Globotriaosylsphingosine, also called lysoglobotriaosylceramide (lysoGb3), is a promising candidate biomarker. METHODS: We synthesized lysoGb3 and isotope-labeled [5,6,7,8,9] (13)C5-lysoGb3 (internal standard). After addition of the internal standard to 25 µL plasma or 400 µL urine from patients with Fabry disease and healthy controls, samples were extracted with organic solvents and the lysoGb3 concentration was quantified by UPLC-ESI-MS/MS (ultraperformance liquid chromatography-electrospray ionization-tandem mass spectrometry). Calibration curves were constructed with control plasma and urine supplemented with lysoGb3. In addition to lysoGb3, lyso-ene-Gb3 was quantified. Quantification was achieved by multiple reaction monitoring of the transitions m/z 786.4 > 282.3 [M+H](+) for lysoGb3, m/z 791.4 > 287.3 [M+H](+) for [5,6,7,8,9] (13)C5-lysoGb3, and 784.4 > 280.3 [M+H](+) for lyso-ene-Gb3. RESULTS: The mean (SD) plasma lysoGb3 concentration from 10 classically affected Fabry hemizygotes was 94.4 (25.8) pmol/mL (range 52.7-136.8 pmol/mL), from 10 classically affected Fabry heterozygotes 9.6 (5.8) pmol/mL (range 4.1-23.5 pmol/mL), and from 20 healthy controls 0.4 (0.1) pmol/mL (range 0.3-0.5 pmol/mL). Lyso-ene-Gb3 concentrations were 10%-25% of total lysoGb3. The urine concentration of lysoGb3 was 40-480 times lower than in corresponding plasma samples. Lyso-ene-Gb3 concentrations in urine were comparable or even higher than the corresponding lysoGb3 concentrations. CONCLUSIONS: This assay for the quantification of lysoGb3 and lyso-ene-Gb3 in human plasma and urine samples will be an important tool in the diagnosis of Fabry disease and for monitoring the effect of enzyme replacement therapy in patients with Fabry disease.

Access to any site directed stable isotope ((2)H, (13)C, (15)N, (17)O and (18)O) in genetically encoded amino acids.

Proteins and peptides play a preeminent role in the processes of living cells. The only way to study structure-function relationships of a protein at the atomic level without any perturbation is by using non-invasive isotope sensitive techniques with site-directed stable isotope incorporation at a predetermined amino acid residue in the protein chain. The method can be extended to study the protein chain tagged with stable isotope enriched amino acid residues at any position or combinations of positions in the system. In order to access these studies synthetic methods to prepare any possible isotopologue and isotopomer of the 22 genetically encoded amino acids have to be available. In this paper the synthetic schemes and the stable isotope enriched building blocks that are available via commercially available stable isotope enriched starting materials are described.

Structure changes upon deprotonation of the proton release group in the bacteriorhodopsin photocycle.

In the photocycle of bacteriorhodopsin at pH 7, a proton is ejected to the extracellular medium during the protonation of Asp-85 upon formation of the M intermediate. The group that releases the ejected proton does not become reprotonated until the prephotolysis state is restored from the N and O intermediates. In contrast, at acidic pH, this proton release group remains protonated to the end of the cycle. Time-resolved Fourier transform infrared measurements obtained at pH 5 and 7 were fitted to obtain spectra of kinetic intermediates, from which the spectra of M and N/O versus unphotolyzed state were calculated. Vibrational features that appear in both M and N/O spectra at pH 7, but not at pH 5, are attributable to deprotonation from the proton release group and resulting structural alterations. Our results agree with the earlier conclusion that this group is a protonated internal water cluster, and provide a stronger experimental basis for this assignment. A decrease in local polarity at the N-C bond of the side chain of Lys-216 resulting from deprotonation of this water cluster may be responsible for the increase in the proton affinity of Asp-85 through M and N/O, which is crucial for maintaining the directionality of proton pumping.

Configuration of spheroidene in the photosynthetic reaction center of Rhodobacter sphaeroides : a comparison of wild-type and reconstituted R26.

We compare the resonance Raman spectra acquired at two excitation wavelengths, 496.5 and 514.5 nm, of spheroidene in the wild-type reaction center of Rhodobacter sphaeroides and reconstituted into the reaction center of the Rhodobacter sphaeroides mutant R26. Our earlier work showed that the reconstituted R26 reaction center holds spheroidene in two configurations: 15,15'-cis and another configuration. Here we show that in the wild-type reaction center only 15,15'-cis spheroidene is present. In the resonance Raman spectra of the reconstituted R26 reaction centers, a transition is identified that arises exclusively from the second configuration. According to density-functional-theory calculations, this transition is specific for the 13,14-cis configuration.

Cyclopropyl and isopropyl derivatives of 11-cis and 9-cis retinals at C-9 and C-13: subtle steric differences with major effects on ligand efficacy in rhodopsin.

Retinal is the natural ligand (chromophore) of the vertebrate rod visual pigment. It occurs in either the 11-cis (rhodopsin) or the 9-cis (isorhodopsin) configuration. In its evolution to a G protein coupled photoreceptor, rhodopsin has acquired exceptional photochemical properties. Illumination isomerizes the chromophore to the all-trans isomer, which acts as a full agonist. This process is extremely efficient, and there is abundant evidence that the C-9 and C-13 methyl groups of retinal play a pivotal role in this process. To examine the steric limits of the C-9 and C-13 methyl binding pocket of the binding site, we have prepared C-9 and C-13 cyclopropyl and isopropyl derivatives of its native ligands and of α-retinal at C-9. Most isopropyl analogues show very poor binding, except for 9-cis-13-isopropylretinal. Most cyclopropyl derivatives exhibit intermediate binding activity, except for 9-cis-13-cyclopropylretinal, which presents good binding activity. In general, the binding site shows preference for the 9-cis analogues over the 11-cis analogues. In fact, 13-isopropyl-9-cis-retinal acts as a superagonist after illumination. Another surprising finding was that 9-cyclopropylisorhodopsin is more like native rhodopsin with respect to spectral and photochemical properties, whereas 9-cyclopropylrhodopsin behaves more like native isorhodopsin in these aspects.

Towards an interpretation of 13C chemical shifts in bathorhodopsin, a functional intermediate of a G-protein coupled receptor.

Photoisomerization of the membrane-bound light receptor protein rhodopsin leads to an energy-rich photostate called bathorhodopsin, which may be trapped at temperatures of 120 K or lower. We recently studied bathorhodopsin by low-temperature solid-state NMR, using in situ illumination of the sample in a purpose-built NMR probe. In this way we acquired (13)C chemical shifts along the retinylidene chain of the chromophore. Here we compare these results with the chemical shifts of the dark state chromophore in rhodopsin, as well as with the chemical shifts of retinylidene model compounds in solution. An earlier solid-state NMR study of bathorhodopsin found only small changes in the (13)C chemical shifts upon isomerization, suggesting only minor perturbations of the electronic structure in the isomerized retinylidene chain. This is at variance with our recent measurements which show much larger perturbations of the (13)C chemical shifts. Here we present a tentative interpretation of our NMR results involving an increased charge delocalization inside the polyene chain of the bathorhodopsin chromophore. Our results suggest that the bathochromic shift of bathorhodopsin is due to modified electrostatic interactions between the chromophore and the binding pocket, whereas both electrostatic interactions and torsional strain are involved in the energy storage mechanism of bathorhodopsin.

Resolution and polarization distribution in cryogenic DNP/MAS experiments.

This contribution addresses four potential misconceptions associated with high-resolution dynamic nuclear polarization/magic angle spinning (DNP/MAS) experiments. First, spectral resolution is not generally compromised at the cryogenic temperatures at which DNP experiments are performed. As we demonstrate at a modest field of 9 T (380 MHz (1)H), 1 ppm linewidths are observed in DNP/MAS spectra of a membrane protein in its native lipid bilayer, and <0.4 ppm linewidths are reported in a crystalline peptide at 85 K. Second, we address the concerns about paramagnetic broadening in DNP/MAS spectra of proteins by demonstrating that the exogenous radical polarizing agents utilized for DNP are distributed in the sample in such a manner as to avoid paramagnetic broadening and thus maintain full spectral resolution. Third, the enhanced polarization is not localized around the polarizing agent, but rather is effectively and uniformly dispersed throughout the sample, even in the case of membrane proteins. Fourth, the distribution of polarization from the electron spins mediated via spin diffusion between (1)H-(1)H strongly dipolar coupled spins is so rapid that shorter magnetization recovery periods between signal averaging transients can be utilized in DNP/MAS experiments than in typical experiments performed at ambient temperature.

Action Spectroscopy on Dense Samples of Photosynthetic Reaction Centers of Rhodobacter sphaeroides WT Based on Nanosecond Laser-Flash C Photo-CIDNP MAS NMR.

Photochemically induced dynamic nuclear polarization magic-angle spinning nuclear magnetic resonance (photo-CIDNP MAS NMR) allows for the investigation of the electronic structure of the photochemical machinery of photosynthetic reaction centers (RCs) at atomic resolution. For such experiments, either continuous radiation from white xenon lamps or green laser pulses are applied to optically dense samples. In order to explore their optical properties, optically thick samples of isolated and quinone-removed RCs of the purple bacteria of Rhodobacter sphaeroides wild type are studied by nanosecond laser-flash (13)C photo-CIDNP MAS NMR using excitation wavelengths between 720 and 940 nm. Action spectra of both the transient nuclear polarization as well as the nuclear hyperpolarization, remaining in the electronic ground state at the end of the photocycle, are obtained. It is shown that the signal intensity is limited by the amount of accessible RCs and that the different mechanisms of the photo-CIDNP production rely on the same photophysical origin, which is the photocycle induced by one single photon.

Synthesis and use of stable isotope enriched retinals in the field of vitamin A.

The role of vitamin A and its metabolites in the life processes starting with the historical background and its up to date information is discussed in the introduction. Also the role of 11Z-retinal in vision and retinoic acid in the biological processes is elucidated. The essential role of isotopically enriched systems in the progress of vision research, nutrition research etc. is discussed. In part B industrial commercial syntheses of vitamin A by the two leading companies Hoffmann-La Roche (now DSM) and BASF are discussed. The knowledge obtained via these pioneering syntheses has been essential for the further synthetic efforts in vitamin A field by other scientific groups. The rest of the paper is devoted to the synthetic efforts of the Leiden group that gives an access to the preparation of site directed high level isotope enrichment in retinals. First the synthesis of the retinals with deuterium incorporation in the conjugated side chain is reviewed. Then, 13C-labeled retinals are discussed. This is followed by the discussion of a convergent synthetic scheme that allows a rational access to prepare any isotopomer of retinals. The schemes that provide access to prepare any possible isotope enriched chemically modified systems are discussed. Finally, nor-retinals and bridged retinals that give access to a whole (as yet incomplete) library of possible isotopomers are reviewed.

Fluoro derivatives of retinal illuminate the decisive role of the C(12)-H element in photoisomerization and rhodopsin activation.

Rhodopsin, the visual pigment of the vertebrate rod cell, is among the best investigated members of the G-protein-coupled receptor family. Within this family a unique characteristic of visual pigments is their covalently bound chromophore, 11-cis retinal, which acts as an inverse agonist. Upon illumination it can be transformed into the all-trans isomer that acts as a full agonist. This photoisomerization process is extremely efficient: 2 out of 3 photons are effective, full stereoselectivity is achieved, and stereoinversion occurs within 200 fs. The mechanism behind this process is still not really understood, although the available evidence points at the twisted C(9)-C(13) segment of the 11-cis ligand as the quintessence. To further dissect the role of this segment, we have generated the 10-fluoro, 12-fluoro, and 14-fluoro analogues of rhodopsin. A fluoro substituent brings in only little more volume than hydrogen, but considerably more mass and polarizability. The analogue pigments were compared to rhodopsin with respect to their photosensitivity (quantum yield), light-induced structural transitions (UV-vis and FT-IR spectroscopy), and signaling activity (G protein activation rate). We find that 14-F substitution is quite neutral, while 10-F and 12-F substitutions exert significant but distinct effects. The 10-F pigment exhibits a quantum yield similar to that of rhodopsin (0.65) but strongly perturbed thermodynamics of the structural transitions following photoactivation and only 20% of the native signaling activity. The 12-F pigment exhibits a significantly decreased quantum yield (0.47) and signaling activity (30%) but mixed effects on the structural transitions. These properties are compared to those of the corresponding methyl derivatives. We conclude that rotation of the C(12)-H bond of the rhodopsin chromophore is a major rate-limiting factor in the photoisomerization process, while the C(10)-H moiety plays a dominant role in ligand relaxation and further rearrangements following photoactivation.

Structural changes due to the deprotonation of the proton release group in the M-photointermediate of bacteriorhodopsin as revealed by time-resolved FTIR spectroscopy.

One of the steps in the proton pumping cycle of bacteriorhodopsin (BR) is the release of a proton from the proton-release group (PRG) on the extracellular side of the Schiff base. This proton release takes place shortly after deprotonation of the Schiff base (L-to-M transition) and results in an increase in the pKa of Asp85, which is a crucial mechanistic step for one-way proton transfer for the entire photocycle. Deprotonation of the PRG can also be brought about without photoactivation, by raising the pH of the enzyme (pKa of PRG; approximately 9). Thus, comparison of the FTIR difference spectrum for formation of the M intermediate (M minus initial unphotolyzed BR state) at pH 7 to the corresponding spectrum generated at pH 10 may reveal structural changes specifically associated with deprotonation of the PRG. Vibrational bands of BR that change upon M formation are distributed across a broad region between 2120 and 1685 cm(-1). This broad band is made up of two parts. The band above 1780 cm(-1), which is insensitive to C15-deuteration of the retinal, may be due to a proton delocalized in the PRG. The band between 1725 and 1685 cm(-1), on the lower frequency side of the broad band, is sensitive to C15-deuteration. This band may arise from transition dipole coupling of the vibrations of backbone carbonyl groups in helix G with the side chain of Tyr57 and with the C15H of the Schiff base. In M, these broad bands are abolished, and the 3657 cm(-1) band, which is due to the disruption of the hydrogen bonding of a water molecule, probably with Arg82, appears. Loss of the interaction of the backbone carbonyl groups in helix G with Tyr57 and the Schiff base, and separation of Tyr57 from Arg82, may be causes of these spectral changes, leading to the stabilization of the protonated Asp85 in M.

Double-quantum 13C nuclear magnetic resonance of bathorhodopsin, the first photointermediate in mammalian vision.

The 13C chemical shifts of the primary visual photointermediate bathorhodopsin have been observed by performing double-quantum magic-angle-spinning NMR at low temperature in the presence of illumination. Strong isomerization shifts have been observed upon the conversion of rhodopsin into bathorhodopsin.

Alpha-retinals as rhodopsin chromophores--preference for the 9-Z configuration and partial agonist activity.

The visual pigment rhodopsin, the photosensory element of the rod photoreceptor cell in the vertebrate retina, shows in combination with an endogenous ligand, 11-Z retinal, an astonishing photochemical performance. It exhibits an unprecedented quantum yield (0.67) in a highly defined and ultrafast photoisomerization process. This triggers the conformational changes leading to the active state Meta(rhodopsin) II. Retinal is covalently bound to Lys-296 of the protein opsin in a protonated Schiff base. The resulting positive charge delocalization over the terminal part of the polyene chain of retinal creates a conjugation defect that upon photoexcitation moves to the opposite end of the polyene. Shortening the polyene as in 4,5-dehydro,5,6-dihydro (alpha), 5,6-dihydro or 7,8-dihydro-analogs might facilitate photoisomerization of a 9-Z and a 11-Z bond. Here we describe pigment analogs generated with bovine opsin and 11-Z or 9-Z 4,5-dehydro,5,6-dihydro-retinal that were further characterized by UV-Vis and FTIR spectroscopy. The preference of opsin for native 11-Z retinal over the 9-Z isomer is reversed in 4,5-dehydro,5,6-dihydro-retinal. 9-Z 4,5-dehydro,5,6-dihydro-retinal readily generated a photosensitive pigment. This modification has no effect on the quantum yield, but affects the Batho<-->blueshifted intermediate (BSI) equilibrium and leads to a strong decrease in the G-protein activation rate because of a downshift of the pK(a) of the Meta I<-->Meta II equilibrium.

The Dynamic Origin of Color Tuning in Proteins Revealed by a Carotenoid Pigment.

Understanding the microscopic origin of the color tuning in pigment-protein complexes is a challenging yet fundamental issue in photoactive biological systems. Here, we propose a possible interpretation by using a state-of-the-art multiscale strategy based on the integration of quantum chemistry and polarizable atomistic embeddings into a dynamic description. By means of such a strategy we are able to resolve the long-standing dispute over the coloration mechanism in the crustacyanin protein. It is shown that the combination of the dynamical flexibility of the carotenoid pigments (astaxanthin) with the responsive protein environment is essential to obtain quantitative predictions of the spectral tuning. The strong linear correlation between the excitation energies and the bond length alternation in the long-chain carotenoids modulated by the dynamical protein environment is a novel finding explaining the high color tunability in crustacyanin.