QM/MM benchmarking of cyanobacteriochrome Slr1393g3 absorption spectra

Cyanobacteriochromes are compact and spectrally diverse photoreceptor proteins that are promising candidates for biotechnological applications. Computational studies can contribute to an understanding at a molecular level of their wide spectral tuning and diversity. In this contribution, we benchmark methods to model a 110 nm shift in the UV/Vis absorption spectrum from a red- to a green-absorbing form of the cyanobacteriochrome Slr1393g3. Based on an assessment of semiempirical methods to describe the chromophore geometries of both forms in vacuo, we find that DFTB2+D leads to structures that are the closest to the reference method. The benchmark of the excited state calculations is based on snapshots from quantum mechanics/molecular mechanics molecular dynamics simulations. In our case, the methods RI-ADC(2) and sTD-DFT based on CAM-B3LYP ground state calculations perform the best, whereas no functional can be recommended to simulate the absorption spectra of both forms with time-dependent density functional theory. Furthermore, the difference in absorption for the lowest energy absorption maxima of both forms can already be modelled with optimized structures, but sampling is required to improve the shape of the absorption bands of both forms, in particular for the second band. This benchmark study can guide further computational studies, as it assesses essential components of a protocol to model the spectral tuning of both cyanobacteriochromes and the related phytochromes

Quantummechanical Investigations of the Photoisomerization of Retinal Model Chromophores

Im Rahmen der vorliegenden Arbeit wurde mit Hilfe quantenmechanischer Methoden die Photoisomerisierung des Retinals simuliert. Sowohl statische als auch dynamische Simulationen wurden durchgeführt, um den Mechanismus der hoch¬effizienten Reaktion auf der molekularen Ebene zu verstehen. Eine Trajektorie des Retinalmodells mit vier Doppelbindungen wurde erhalten, in der die beiden mittleren Doppelbindungen ähnlich wie im Bicycle-Pedal-Mechanismus isomerisierten. Außerdem wurde festgestellt, dass es zwei Permu¬tationen gibt für den Rotationssinn der isomerisierenden Doppelbindungen: in die gleiche Richtung, konrotatorisch, oder in die entgegengesetzte Richtung, also dis¬rotatorisch. Relaxierte Pfade haben schließlich gezeigt, dass beide Möglichkeiten keine Energiebarriere haben und realisiert werden können. Allerdings wurde keine Trajektorie gefunden mit der disrotatorischen Rotation der Doppelbindungen. Mit Hilfe dieser statischen Berechnungen mit festgehaltenen Diederwinkeln konnte eine Vielfalt von Reaktionspfaden für das Chromophormodell unter isolierten Bedingungen demonstriert werden. Mittels dynamischer Simulationen wurden im nächsten Abschnitt Produktausbeuten und Verweilzeiten im angeregten Zustand bestimmt. Dazu wurde das im Rahmen dieser Arbeit entwickelte und in MOLCAS-Programmpaket implementierte Modul DYNAMIX verwendet. Drei Gruppen von Fünfdoppelbindungsmodellen wurden untersucht. Die erste Gruppe bestand aus den vier Isomeren 9-cis, 11-cis, 13-cis und all-trans. Es wurde gezeigt, dass bereits im Vakuum das 11-cis-Isomer eine erhöhte Selektivität aufzeigt und ausschließlich um die cis-Bindung rotiert. Auch bei den Quantenausbeuten wurde eine grobe Übereinstimmung mit den entsprechenden Retinal-Isomeren eingebettet im Opsin gefunden. Daraus folgt, dass sowohl die Selektivität als auch die Effizienz der Retinalisomerisierung eine intrinsische Eigenschaft ist, d.h. bereits ohne Protein vorhanden ist. Sechs verschiedene methylsubstituierte Chromophore bildeten die zweite Gruppe von Modellen. Durch gezielte Manipulation der intramolekularen sterischen Wechselwirkungen konnten die Quantenausbeute, die Selektivität des Produktes und die Reaktionszeit abgestimmt werden. Durch eine zusätzliche Methylgruppe am C10-Kohlenstoffatom konnte beispielsweise die Reaktionszeit drastisch verkürzt und eine unidirektionale Isomerisierung erzwungen werden. Die Substitution aller Methyl¬gruppen durch Wasserstoffe führt zur vollständigen Umsetzung zum trans-Produkt. Diese Erkenntnisse sind besonders von hohem Interesse, um künstliche Photo-Schalter mit maßgeschneiderten Eigenschaften zu entwerfen. Die letzte Gruppe umfasste drei 11-cis-verbrückte Modellchromophore bestehend aus einem 5-, 7- und 8-Ring. Der kleinste Ring zeigte keine Tendenz zur Photoisomerisierung und alle Trajektorien verblieben während der gesamten Simulationszeit im angeregten Zustand. Beim siebengliedrigen Ring wurden Isomerisierungen um die drei zentralen Doppelbindungen beobachtet: C9-C10, C11-C12 und C13-C14. Am häufigsten wurde die Isomerisierung um die Bindung C9-C10, die außerhalb des Rings liegt, beobachtet. Durch die Erweiterung des Ringes um eine Methyleneinheit wird eine größere Flexibilität erreicht, sodass im 8-Ring alle Isomerisierungen um die im Ring liegenden Doppelbindungen C11-C12 ablaufen. Die Verweildauer im angeregten Zustand wird deutlich kürzer, da der Ring eine Vorverdrillung der C11-C12 Bindung mit sich bringt. Die beiden letztgenannten Modelle haben gemeinsam, dass der Rotationssinn durch die Ringkonformation bestimmt wird. Damit wird ein ähnlicher Effekt erreicht wie die Methylsubstitution, die in einigen Retinalmodellen zu erhöhter sterischer Wechselwirkung führt. Im letzten Abschnitt der Arbeit wurde mit QM/MM MD Simulationen der Einfluss der Proteinumgebung auf die Photoisomerisierung der Retinals untersucht. Aufgrund des enormen Zeitaufwands wurde jeweils eine Trajektorie für drei Photopigmente berechnet, nämlich: Rhodopsin, Bathorhodopsin und Isorhodopsin. Zwischen den Trajektorien und den experimentellen Untersuchungen wurde eine Korrelation gefunden. So entspricht die relative Verweilzeit im angeregten Zustand den Ergebnissen aus zeitaufgelösten Transienten-Absorptionsspektroskopie Messungen. Ebenfalls sind die erfolgreichen Isomerisierungen von Rhodopsin und Bathorhodopsin sowie die gescheiterte Isomerisierung des Isorhodopsins konsistent mit experimentell bestimmten Quantenausbeuten. Die Photoisomerisierung des Rhodopsins und des Bathorhodopsins erfolgt nach dem abgebrochenen Bicycle-Pedal-Mechanismus, bei dem die Rotation um die C11-C12 Bindung durch die Rotation der beiden benachbarten Doppelbindungen unterstützt wird. Dadurch wird eine raumsparende sowie ultraschnelle Isomerisierung des Retinals ermöglicht

Ultrafast Backbone Protonation in Channelrhodopsin-1 Captured by Polarization Resolved Fs Vis-pump - IR-Probe Spectroscopy and Computational Methods

Channelrhodopsins (ChR) are light-gated ion-channels heavily used in optogenetics. Upon light excitation an ultrafast all-trans to 13-cis isomerization of the retinal chromophore takes place. It is still uncertain by what means this reaction leads to further protein changes and channel conductivity. Channelrhodopsin-1 in Chlamydomonas augustae exhibits a 100 fs photoisomerization and a protonated counterion complex. By polarization resolved ultrafast spectroscopy in the mid-IR we show that the initial reaction of the retinal is accompanied by changes in the protein backbone and ultrafast protonation changes at the counterion complex comprising Asp299 and Glu169. In combination with homology modelling and quantum mechanics/molecular mechanics (QM/MM) geometry optimization we assign the protonation dynamics to ultrafast deprotonation of Glu169, and transient protonation of the Glu169 backbone, followed by a proton transfer from the backbone to the carboxylate group of Asp299 on a timescale of tens of picoseconds. The second proton transfer is not related to retinal dynamics and reflects pure protein changes in the first photoproduct. We assume these protein dynamics to be the first steps in a cascade of protein-wide changes resulting in channel conductivit

Atomistic Insight into the Role of Threonine 127 in the Functional Mechanism of Channelrhodopsin-2

Channelrhodopsins (ChRs) belong to the unique class of light-gated ion channels. The structure of channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) has been resolved, but the mechanistic link between light-induced isomerization of the chromophore retinal and channel gating remains elusive. Replacements of residues C128 and D156 (DC gate) resulted in drastic effects in channel closure. T127 is localized close to the retinal Schiff base and links the DC gate to the Schiff base. The homologous residue in bacteriorhodopsin (T89) has been shown to be crucial for the visible absorption maximum and dark–light adaptation, suggesting an interaction with the retinylidene chromophore, but the replacement had little effect on photocycle kinetics and proton pumping activity. Here, we show that the T127A and T127S variants of CrChR2 leave the visible absorption maximum unaffected. We inferred from hybrid quantum mechanics/molecular mechanics (QM/MM) calculations and resonance Raman spectroscopy that the hydroxylic side chain of T127 is hydrogen-bonded to E123 and the latter is hydrogen-bonded to the retinal Schiff base. The C=N–H vibration of the Schiff base in the T127A variant was 1674 cm−1, the highest among all rhodopsins reported to date. We also found heterogeneity in the Schiff base ground state vibrational properties due to different rotamer conformations of E123. The photoreaction of T127A is characterized by a long-lived P2380 state during which the Schiff base is deprotonated. The conservative replacement of T127S hardly affected the photocycle kinetics. Thus, we inferred that the hydroxyl group at position 127 is part of the proton transfer pathway from D156 to the Schiff base during rise of the P3530 intermediate. This finding provides molecular reasons for the evolutionary conservation of the chemically homologous residues threonine, serine, and cysteine at this position in all channelrhodopsins known so far

Transient 2D IR spectroscopy and multiscale simulations reveal vibrational couplings in the Cyanobacteriochrome Slr1393-g3

Cyanobacteriochromes are bi-stable photoreceptor proteins with desirable photochemical properties for biotechnological applications such as optogenetics or fluorescence microscopy. Here, we investigated Slr1393-g3, a cyanobacteriochrome that reversibly photo-switches between a red-absorbing (Pr) and green-absorbing (Pg) form. We applied advanced IR spectroscopic methods to track the sequence of intermediates during the photocycle over many orders in magnitude in time. In the conversion from Pg to Pr, we have revealed a new intermediate which precedes the Pr formation by using transient IR spectroscopy. In addition, stationary and transient 2D~IR experiments measured the vibrational couplings between different groups of the chromophore and the protein during these intermediate states. Anharmonic QM/MM calculations predict spectra in close-to-quantitative agreement with experimental 2D~IR spectra of the initial and the final state of the photocycle. They facilitate the assignment of the IR spectra and provide an atomistic insight into the coupling mechanism. This serves as a basis for the interpretation of the spectroscopic results and suggests structural changes of the intermediates along the photocycle

Retinal chromophore charge delocalization and confinement explain the extreme photophysics of Neorhodopsin

The understanding of how the rhodopsin sequence can be modified to exactly modulate the spectroscopic properties of its retinal chromophore, is a prerequisite for the rational design of more effective optogenetic tools. One key problem is that of establishing the rules to be satisfied for achieving highly fluorescent rhodopsins with a near infrared absorption. In the present paper we use multi-configurational quantum chemistry to construct a computer model of a recently discovered natural rhodopsin, Neorhodopsin, displaying exactly such properties. We show that the model, that successfully replicates the relevant experimental observables, unveils a geometrical and electronic structure of the chromophore featuring a highly diffuse charge distribution along its conjugated chain. The same model reveals that a charge confinement process occurring along the chromophore excited state isomerization coordinate, is the primary cause of the observed fluorescence enhancement

Assessment Of Density Functional Theory For Describing The Correlation Effects On The Ground And Excited State Potential Energy Surfaces Of A Retinal Chromophore Model

In the quest for a cost-effective level of theory able to describe a large portion of the ground and excited potential energy surfaces of large chromophores, promising approaches are rooted in various approximations to the exact density functional theory (DFT). In the present work, we investigate how generalized Kohn-Sham DFT (GKS-DFT), time-dependent DFT (TDDFT), and spin-restricted ensemble-DFT (REKS) methods perform along three important paths characterizing a model retinal chromophore (the penta-2,4-dieniminium cation) in a region of near-degeneracy (close to a conical intersection) with respect to reference high-level multiconfigurational wave function methods. If GKS-DFT correctly describes the closed-shell charge transfer state, only TDDFT and REKS approaches give access to the open-shell diradical, one which sometimes corresponds to the electronic ground state. It is demonstrated that the main drawback of the usual DFT-based methods lies in the absence of interactions between the charge transfer and the diradicaloid configurations. Hence, we test a new computational scheme based on the State-averaged REKS (SA-REKS) approach, which explicitly includes these interactions into account. The State-Interaction SA-REKS (SI-SA-REKS) method significantly improves on the REKS and the SA-REKS results for the target system. The similarities and differences between DFT and wave function-based approaches are analyzed according to (1) the active space dimensions of the wave function-based methods and (2) the relative electronegativities of the allyl and protonated Schiff base moieties

Structural elements regulating the photochromicity in a cyanobacteriochrome

The three-dimensional (3D) crystal structures of the GAF3 domain of cyanobacteriochrome Slr1393 (Synechocystis PCC6803) carrying a phycocyanobilin chromophore could be solved in both 15-Z dark-adapted state, Pr, λmax = 649 nm, and 15-E photoproduct, Pg, λmax = 536 nm (resolution, 1.6 and 1.86 Å, respectively). The structural data allowed identifying the large spectral shift of the Pr-to-Pg conversion as resulting from an out-of-plane rotation of the chromophore’s peripheral rings and an outward movement of a short helix formed from a formerly unstructured loop. In addition, a third structure (2.1-Å resolution) starting from the photoproduct crystals allowed identification of elements that regulate the absorption maxima. In this peculiar form, generated during X-ray exposition, protein and chromophore conformation still resemble the photoproduct state, except for the D-ring already in 15-Z configuration and tilted out of plane akin the dark state. Due to its formation from the photoproduct, it might be considered an early conformational change initiating the parental state-recovering photocycle. The high quality and the distinct features of the three forms allowed for applying quantum-chemical calculations in the framework of multiscale modeling to rationalize the absorption maxima changes. A systematic analysis of the PCB chromophore in the presence and absence of the protein environment showed that the direct electrostatic effect is negligible on the spectral tuning. However, the protein forces the outer pyrrole rings of the chromophore to deviate from coplanarity, which is identified as the dominating factor for the color regulation

Structure Of The Photochemical Reaction Path Populated Via Promotion Of Cf2i2 Into Its First Excited State

The photochemical reaction path following the promotion of CF2I2 into its lowest-lying excited electronic singlet state has been modeled using ab initio multiconfigurational quantum chemical calculations. It is found that a conical intersection drives the electronically excited CF2I2* species either to the CF2I + I radical pair or back to the starting CF2I2 structure. The structures of the computed relaxation pathways explain the photoproduct selectivity previously observed in the gas phase. Furthermore, the results provide the basis for explaining the condensed-phase photochemistry of CF2I2

The Ultrafast Photoisomerizations Of Rhodopsin And Bathorhodopsin Are Modulated By Bond Length Alternation And Hoop Driven Electronic Effects

Rhodopsin (Rh) and bathorhodopsin (bathoRh) quantum-mechanics/molecular-mechanics models based on ab initio multiconfigurational wave functions are employed to look at the light induced pi-bond breaking and reconstitution occurring during the Rh -\u3e bathoRh and bathoRh -\u3e Rh isomerizations. More specifically, semiclassical trajectory computations are used to compare the excited (S-1) and ground (S-0) state dynamics characterizing the opposite steps of the Rh/bathoRh photochromic cycle during the first 200 fs following photoexcitation. We show that the information contained in these data provide an unprecedented insight into the sub-picosecond pi-bond reconstitution process which is at the basis of the reactivity of the protein embedded 11-cis and all-trans retinal chromophores. More specifically, the data point to the phase and amplitude of the skeletal bond length alternation stretching mode as the key factor switching the chromophore to a bonding state. It is also confirmed/found that the phase and amplitude of the hydrogen-out-of-plane mode controls the stereochemical outcome of the forward and reverse photoisomerizations