Spectroscopic and Computational Observation of Glutamine Tautomerization in the Blue Light Sensing Using Flavin Domain Photoreaction.

Blue light sensing using flavin (BLUF) domains constitute a family of flavin-binding photoreceptors of bacteria and eukaryotic algae. BLUF photoactivation proceeds via a light-driven hydrogen-bond switch among flavin adenine dinucleotide (FAD) and glutamine and tyrosine side chains, whereby FAD undergoes electron and proton transfer with tyrosine and is subsequently re-oxidized by a hydrogen back-shuttle in picoseconds, constituting an important model system to understand proton-coupled electron transfer in biology. The specific structure of the hydrogen-bond patterns and the prevalence of glutamine tautomeric states in dark-adapted (DA) and light-activated (LA) states have remained controversial. Here, we present a combined femtosecond stimulated Raman spectroscopy (FSRS), computational chemistry, and site-selective isotope labeling Fourier-transform infrared spectroscopy (FTIR) study of the Slr1694 BLUF domain. FSRS showed distinct vibrational bands from the FADS1 singlet excited state. We observed small but significant shifts in the excited-state vibrational frequency patterns of the DA and LA states, indicating that these frequencies constitute a sensitive probe for the hydrogen-bond arrangement around FAD. Excited-state model calculations utilizing four different realizations of hydrogen bond patterns and glutamine tautomeric states were consistent with a BLUF reaction model that involved glutamine tautomerization to imidic acid, accompanied by a rotation of its side chain. A combined FTIR and double-isotope labeling study, with 13C labeling of FAD and 15N labeling of glutamine, identified the glutamine imidic acid C═N stretch vibration in the LA state and the Gln C═O in the DA state. Hence, our study provides support for glutamine tautomerization and side-chain rotation in the BLUF photoreaction.

Fentanyl and the Fluorinated Fentanyl Derivative NFEPP Elicit Distinct Hydrogen-Bond Dynamics of the Opioid Receptor.

The development of safe therapeutics to manage pain is of central interest for biomedical applications. The fluorinated fentanyl derivative N-(3-fluoro-1-phenethylpiperidin-4-yl)-N-phenylpropionamide (NFEPP) is potentially a safer alternative to fentanyl because unlike fentanyl─which binds to the µ-opioid receptor (MOR) at both physiological and acidic pH─NFEPP might bind to the MOR only at acidic pH typical of inflamed tissue. Knowledge of the protonation-coupled dynamics of the receptor-drug interactions is thus required to understand the molecular mechanism by which receptor activation initiates cell signaling to silence pain. To this end, here we have carried out extensive atomistic simulations of the MOR in different protonation states, in the absence of opioid drugs, and in the presence of fentanyl vs NFEPP. We used graph-based analyses to characterize internal hydrogen-bond networks that could contribute to the activation of the MOR. We find that fentanyl and NFEPP prefer distinct binding poses and that, in their binding poses, fentanyl and NFEPP partake in distinct internal hydrogen-bond networks, leading to the cytoplasmic G-protein-binding region. Moreover, the protonation state of functionally important aspartic and histidine side chains impacts hydrogen-bond networks that extend throughout the receptor, such that the ligand-bound MOR presents at its cytoplasmic G-protein-binding side, a hydrogen-bonding environment where dynamics depend on whether fentanyl or NFEPP is bound, and on the protonation state of specific MOR groups. The exquisite sensitivity of the internal protein-water hydrogen-bond network to the protonation state and to details of the drug binding could enable the MOR to elicit distinct pH- and opioid-dependent responses at its cytoplasmic G-protein-binding site.

Quantum-mechanical insights into the anisotropic response of the cryptochrome radical pair to a weak magnetic field.

Cryptochrome photoreceptors contain a photochemically generated radical pair, which is thought to mediate sensing of the geomagnetic field direction in many living organisms. To gain insight into the response of the cryptochrome to a weak magnetic field, we have studied the quantum-mechanical hyperfine spin states of the radical pair. We identify quantum states responsible for the precise detection of the magnetic field direction, taking into account the strongly axial hyperfine interactions of each radical in the radical pair. The contribution of these states to the formation of the cryptochrome signaling state sharply increases when the magnetic field becomes orthogonal to the hyperfine axis of either radical. Due to such a response, the radical pair may be able to detect the particular field direction normal to the plane containing the hyperfine axes of the radicals.

QM/MM Modeling of the Flavin Functionalization in the RutA Monooxygenase.

Oxygenase activity of the flavin-dependent enzyme RutA is commonly associated with the formation of flavin-oxygen adducts in the enzyme active site. We report the results of quantum mechanics/molecular mechanics (QM/MM) modeling of possible reaction pathways initiated by various triplet state complexes of the molecular oxygen with the reduced flavin mononucleotide (FMN) formed in the protein cavities. According to the calculation results, these triplet-state flavin-oxygen complexes can be located at both re-side and si-side of the isoalloxazine ring of flavin. In both cases, the dioxygen moiety is activated by electron transfer from FMN, stimulating the attack of the arising reactive oxygen species at the C4a, N5, C6, and C8 positions in the isoalloxazine ring after the switch to the singlet state potential energy surface. The reaction pathways lead to the C(4a)-peroxide, N(5)-oxide, or C(6)-hydroperoxide covalent adducts or directly to the oxidized flavin, depending on the initial position of the oxygen molecule in the protein cavities.

Rational Control of Off-State Heterogeneity in a Photoswitchable Fluorescent Protein Provides Switching Contrast Enhancement.

Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, one of the key parameters that dictate the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.

CHARMM Force-Field Parameters for Morphine, Heroin, and Oliceridine, and Conformational Dynamics of Opioid Drugs.

Opioid drug binding to specialized G protein-coupled receptors (GPCRs) can lead to analgesia upon activation via downstream Gi protein signaling and to severe side effects via activation of the ß-arrestin signaling pathway. Knowledge of how different opioid drugs interact with receptors is essential, as it can inform and guide the design of safer therapeutics. We performed quantum and classical mechanical computations to explore the potential energy landscape of four opioid drugs: morphine and its derivatives heroin and fentanyl and for the unrelated oliceridine. From potential energy profiles for bond twists and from interactions between opioids and water, we derived a set of force-field parameters that allow a good description of structural properties and intermolecular interactions of the opioids. Potential of mean force profiles computed from molecular dynamics simulations indicate that fentanyl and oliceridine have complex energy landscapes with relatively small energy penalties, suggesting that interactions with the receptor could select different binding poses of the drugs.

Four resonance structures elucidate double-bond isomerisation of a biological chromophore.

Photoinduced double-bond isomerisation of the chromophore of photoactive yellow protein (PYP) is highly sensitive to chromophore-protein interactions. On the basis of high-level ab initio calculations, we scrutinise the effect of hydrogen bonds on the photophysical and photochemical properties of the chromophore. We identify four resonance structures - two closed-shell and two biradicaloid - that elucidate the electronic structure of the ground and first excited states involved in the isomerisation process. Changing the relative energies of the resonance structures by hydrogen-bonding interactions tunes all photochemical properties of the chromophore in an interdependent manner. Our study sheds new light on the role of the chromophore electronic structure in tuning in photosensors and fluorescent proteins.

Spiers Memorial Lecture. Introductory lecture: the impact of structure on photoinduced processes in nucleic acids and proteins.

Light is an important environmental variable and most organisms have evolved means to sense, exploit or avoid it and to repair detrimental effects on their genome. In general, light absorption is the task of specific chromophores, however other biomolecules such as oligonucleotides also do so which can result in undesired outcomes such as mutations and cancer. Given the biological importance of light-induced processes and applications for imaging, optogenetics, photodynamic therapy or photovoltaics, there is a great interest in understanding the detailed molecular mechanisms of photoinduced processes in proteins and nucleic acids. The processes are typically characterized by time-resolved spectroscopic approaches or computation, inferring structural information on transient species from stable ground state structures. Recently, however, structure determination of excited states or other short-lived species has become possible with the advent of X-ray free-electron lasers. This review gives an overview of the impact of structure on the understanding of photoinduced processes in macromolecules, focusing on systems presented at this Faraday Discussion meeting.

Indications of 5' to 3' Interbase Electron Transfer as the First Step of Pyrimidine Dimer Formation Probed by a Dinucleotide Analog.

Pyrimidine dimers are the most common DNA lesions generated under UV radiation. To reveal the molecular mechanisms behind their formation, it is of significance to reveal the roles of each pyrimidine residue. We thus replaced the 5'-pyrimidine residue with a photochemically inert xylene moiety (X). The electron-rich X can be readily oxidized but not reduced, defining the direction of interbase electron transfer (ET). Irradiation of the XpT dinucleotide under 254 nm UV light generates two major photoproducts: a pyrimidine (6-4) pyrimidone analog (6-4PP) and an analog of the so-called spore photoproduct (SP). Both products are formed by reaction at C4=O of the photo-excited 3'-thymidine (T), which indicates that excitation of a single "driver" residue is sufficient to trigger pyrimidine dimerization. Our quantum-chemical calculations demonstrated that photo-excited 3'-T accepts an electron from 5'-X. The resulting charge-separated radical pair lowers its energy upon formation of interbase covalent bonds, eventually yielding 6-4PP and SP.

Single Hydrogen Bond Donation from Flavin N5 to Proximal Asparagine Ensures FAD Reduction in DNA Photolyase.

The spread of the absorbance of the stable FADH(•) radical (300-700 nm) allows CPD photolyase to highly efficiently form FADH(-), making it functional for DNA repair. In this study, FTIR spectroscopy detected a strong hydrogen bond, from FAD N5-H to the carbonyl group of the Asn378 side chain, that is modulated by the redox state of FAD. The observed characteristic frequency shifts were reproduced in quantum-mechanical models of the flavin binding site, which were then employed to elucidate redox tuning governed by Asn378. We demonstrate that enhanced hydrogen bonding of the Asn378 side chain with the FADH(•) radical increases thermodynamic stabilization of the radical state, and further ensures kinetic stabilization and accumulation of the fully reduced FADH(-) state.

Quantum-based Modeling of Protein-ligand Interaction: The Complex of RutA with Uracil and Molecular Oxygen.

Modern quantum-based methods are employed to model interaction of the flavin-dependent enzyme RutA with the uracil and oxygen molecules. This complex presents the structure of reactants for the chain of chemical reactions of monooxygenation in the enzyme active site, which is important in drug metabolism. In this case, application of quantum-based approaches is an essential issue, unlike conventional modeling of protein-ligand interaction with force fields using molecular mechanics and classical molecular dynamics methods. We focus on two difficult problems to characterize the structure of reactants in the RutA-FMN-O2 -uracil complex, where FMN stands for the flavin mononucleotide species. First, location of a small O2 molecule in the triplet spin state in the protein cavities is required. Second, positions of both ligands, O2 and uracil, must be specified in the active site with a comparable accuracy. We show that the methods of molecular dynamics with the interaction potentials of quantum mechanics/molecular mechanics theory (QM/MM MD) allow us to characterize this complex and, in addition, to surmise possible reaction mechanism of uracil oxygenation by RutA.

Decrypting cryptochrome: revealing the molecular identity of the photoactivation reaction.

Migrating birds fly thousands of miles or more, often without visual cues and in treacherous winds, yet keep direction. They employ for this purpose, apparently as a powerful navigational tool, the photoreceptor protein cryptochrome to sense the geomagnetic field. The unique biological function of cryptochrome supposedly arises from a photoactivation reaction involving radical pair formation through electron transfer. Radical pairs, indeed, can act as a magnetic compass; however, the cryptochrome photoreaction pathway is not fully resolved yet. To reveal this pathway and underlying photochemical mechanisms, we carried out a combination of quantum chemical calculations and molecular dynamics simulations on plant ( Arabidopsis thaliana ) cryptochrome. The results demonstrate that after photoexcitation a radical pair forms, becomes stabilized through proton transfer, and decays back to the protein's resting state on time scales allowing the protein, in principle, to act as a radical pair-based magnetic sensor. We briefly relate our findings on A. thaliana cryptochrome to photoreaction pathways in animal cryptochromes.

Insight into the structural dynamics of light sensitive proteins from time-resolved crystallography and quantum chemical calculations.

The structural dynamics underlying molecular mechanisms of light-sensitive proteins can be studied by a variety of experimental and computational biophysical techniques. Here we review recent progress in combining time-resolved crystallography at X-ray free electron lasers and quantum chemical calculations to study structural changes in photoenzymes, photosynthetic proteins, photoreceptors, and photoswitchable fluorescent proteins following photoexcitation.

Neutral histidine and photoinduced electron transfer in DNA photolyases.

The two major UV-induced DNA lesions, the cyclobutane pyrimidine dimers (CPD) and (6-4) pyrimidine-pyrimidone photoproducts, can be repaired by the light-activated enzymes CPD and (6-4) photolyases, respectively. It is a long-standing question how the two classes of photolyases with alike molecular structure are capable of reversing the two chemically different DNA photoproducts. In both photolyases the repair reaction is initiated by photoinduced electron transfer from the hydroquinone-anion part of the flavin adenine dinucleotide (FADH(-)) cofactor to the photoproduct. Here, the state-of-the-art XMCQDPT2-CASSCF approach was employed to compute the excitation spectra of the respective active site models. It is found that protonation of His365 in the presence of the hydroquinone-anion electron donor causes spontaneous, as opposed to photoinduced, coupled proton and electron transfer to the (6-4) photoproduct. The resulting neutralized biradical, containing the neutral semiquinone and the N3'-protonated (6-4) photoproduct neutral radical, corresponds to the lowest energy electronic ground-state minimum. The high electron affinity of the N3'-protonated (6-4) photoproduct underlines this finding. Thus, it is anticipated that the (6-4) photoproduct repair is assisted by His365 in its neutral form, which is in contrast to the repair mechanisms proposed in the literature. The repair via hydroxyl group transfer assisted by neutral His365 is considered. The repair involves the 5'base radical anion of the (6-4) photoproduct which in terms of electronic structure is similar to the CPD radical anion. A unified model of the CPD and (6-4) photoproduct repair is proposed.

X-ray and NMR crystallography in an enzyme active site: the indoline quinonoid intermediate in tryptophan synthase.

Chemical-level details such as protonation and hybridization state are critical for understanding enzyme mechanism and function. Even at high resolution, these details are difficult to determine by X-ray crystallography alone. The chemical shift in NMR spectroscopy, however, is an extremely sensitive probe of the chemical environment, making solid-state NMR spectroscopy and X-ray crystallography a powerful combination for defining chemically detailed three-dimensional structures. Here we adopted this combined approach to determine the chemically rich crystal structure of the indoline quinonoid intermediate in the pyridoxal-5'-phosphate-dependent enzyme tryptophan synthase under conditions of active catalysis. Models of the active site were developed using a synergistic approach in which the structure of this reactive substrate analogue was optimized using ab initio computational chemistry in the presence of side-chain residues fixed at their crystallographically determined coordinates. Various models of charge and protonation state for the substrate and nearby catalytic residues could be uniquely distinguished by their calculated effects on the chemical shifts measured at specifically (13)C- and (15)N-labeled positions on the substrate. Our model suggests the importance of an equilibrium between tautomeric forms of the substrate, with the protonation state of the major isomer directing the next catalytic step.

Protonation States of Molecular Groups in the Chromophore-Binding Site Modulate Properties of the Reversibly Switchable Fluorescent Protein rsEGFP2.

The role of protonation states of the chromophore and its neighboring amino acid side chains of the reversibly switching fluorescent protein rsEGFP2 upon photoswitching is characterized by molecular modeling methods. Numerous conformations of the chromophore-binding site in computationally derived model systems are obtained using the quantum chemistry and QM/MM approaches. Excitation energies are computed using the extended multiconfigurational quasidegenerate perturbation theory (XMCQDPT2). The obtained structures and absorption spectra allow us to provide an interpretation of the observed structural and spectral properties of rsEGFP2 in the active ON and inactive OFF states. The results demonstrate that in addition to the dominating anionic and neutral forms of the chromophore, the cationic and zwitterionic forms may participate in the photoswitching of rsEGFP2. Conformations and protonation forms of the Glu223 and His149 side chains in the chromophore-binding site play an essential role in stabilizing specific protonation forms of the chromophore.

Frontiers in Multiscale Modeling of Photoreceptor Proteins.

This perspective article highlights the challenges in the theoretical description of photoreceptor proteins using multiscale modeling, as discussed at the CECAM workshop in Tel Aviv, Israel. The participants have identified grand challenges and discussed the development of new tools to address them. Recent progress in understanding representative proteins such as green fluorescent protein, photoactive yellow protein, phytochrome, and rhodopsin is presented, along with methodological developments.

Primary reactions of the LOV2 domain of phototropin studied with ultrafast mid-infrared spectroscopy and quantum chemistry.

Phototropins, major blue-light receptors in plants, are sensitive to blue light through a pair of flavin mononucleotide (FMN)-binding light oxygen and voltage (LOV) domains, LOV1 and LOV2. LOV2 undergoes a photocycle involving light-driven covalent adduct formation between a conserved cysteine and the FMN C(4a) atom. Here, the primary reactions of Avena sativa phototropin 1 LOV2 (AsLOV2) were studied using ultrafast mid-infrared spectroscopy and quantum chemistry. The singlet excited state (S1) evolves into the triplet state (T1) with a lifetime of 1.5 ns at a yield of approximately 50%. The infrared signature of S1 is characterized by absorption bands at 1657 cm(-1), 1495-1415 cm(-1), and 1375 cm(-1). The T1 state shows infrared bands at 1657 cm(-1), 1645 cm(-1), 1491-1438 cm(-1), and 1390 cm(-1). For both electronic states, these bands are assigned principally to C=O, C=N, C-C, and C-N stretch modes. The overall downshifting of C=O and C=N bond stretch modes is consistent with an overall bond-order decrease of the conjugated isoalloxazine system upon a pi-pi* transition. The configuration interaction singles (CIS) method was used to calculate the vibrational spectra of the S1 and T1 excited pipi* states, as well as respective electronic energies, structural parameters, electronic dipole moments, and intrinsic force constants. The harmonic frequencies of S1 and T1, as calculated by the CIS method, are in satisfactory agreement with the evident band positions and intensities. On the other hand, CIS calculations of a T1 cation that was protonated at the N(5) site did not reproduce the experimental FMN T1 spectrum. We conclude that the FMN T1 state remains nonprotonated on a nanosecond timescale, which rules out an ionic mechanism for covalent adduct formation involving cysteine-N(5) proton transfer on this timescale. Finally, we observed a heterogeneous population of singly and doubly H-bonded FMN C(4)=O conformers in the dark state, with stretch frequencies at 1714 cm(-1) and 1694 cm(-1), respectively.

Electronic structure of (6-4) DNA photoproduct repair involving a non-oxetane pathway.

Mutagenic pyrimidine-pyrimidone (6-4) photoproducts are one of the main DNA lesions induced by solar UV radiation. These lesions can be photoreversed by (6-4) photolyases. The originally published repair mechanism involves rearrangement of the lesion into an oxetane intermediate upon binding to the (6-4) photolyase, followed by light-induced electron transfer from the reduced flavin cofactor. In a recent crystallographic study on a (6-4) photoproduct complexed with (6-4) photolyase from Drosophila melanogaster no oxetane was observed, raising the possibility of a non-oxetane repair mechanism. Using quantum-chemical calculations we find that in addition to repair via an oxetane, a direct transfer of the hydroxyl group results in reversal of the radical anion (6-4) photoproduct. In both mechanisms, the transition states have high energies and correspond to avoided crossings of the ground and excited electronic states. To study whether the repair can proceed via these state crossings, the excited-state potential energy curves were computed. The radical excitation energies and accessibility of the nonadiabatic repair path were found to depend on hydrogen bonds and the protonation state of the lesion. On the basis of the energy calculations, a nonadiabatic repair of the excited (6-4) lesion radical anion via hydroxyl transfer is probable. This repair mechanism is in line with the recent structural data on the (6-4) photolyase from D. melanogaster .

Three-dimensional view of ultrafast dynamics in photoexcited bacteriorhodopsin.

Bacteriorhodopsin (bR) is a light-driven proton pump. The primary photochemical event upon light absorption is isomerization of the retinal chromophore. Here we used time-resolved crystallography at an X-ray free-electron laser to follow the structural changes in multiphoton-excited bR from 250 femtoseconds to 10 picoseconds. Quantum chemistry and ultrafast spectroscopy were used to identify a sequential two-photon absorption process, leading to excitation of a tryptophan residue flanking the retinal chromophore, as a first manifestation of multiphoton effects. We resolve distinct stages in the structural dynamics of the all-trans retinal in photoexcited bR to a highly twisted 13-cis conformation. Other active site sub-picosecond rearrangements include correlated vibrational motions of the electronically excited retinal chromophore, the surrounding amino acids and water molecules as well as their hydrogen bonding network. These results show that this extended photo-active network forms an electronically and vibrationally coupled system in bR, and most likely in all retinal proteins.