A Synthetic Oxygen Sensor for Plants Based on Animal Hypoxia Signaling.

Due to the involvement of oxygen in many essential metabolic reactions, all living organisms have developed molecular systems that allow adaptive physiological and metabolic transitions depending on oxygen availability. In mammals, the expression of hypoxia-response genes is controlled by the heterodimeric Hypoxia-Inducible Factor. The activity of this transcriptional regulator is linked mainly to the oxygen-dependent hydroxylation of conserved proline residues in its α-subunit, carried out by prolyl-hydroxylases, and subsequent ubiquitination via the E3 ligase von Hippel-Lindau tumor suppressor, which targets Hypoxia-Inducible Factor-α to the proteasome. By exploiting bioengineered versions of this mammalian oxygen sensor, we designed and optimized a synthetic device that drives gene expression in an oxygen-dependent fashion in plants. Transient assays in Arabidopsis (Arabidopsis thaliana) mesophyll protoplasts indicated that a combination of the yeast Gal4/upstream activating sequence system and the mammalian oxygen sensor machinery can be used effectively to engineer a modular, oxygen-inducible transcriptional regulator. This synthetic device also was shown to be selectively controlled by oxygen in whole plants when its components were expressed stably in Arabidopsis seedlings. We envision the exploitation of our genetically encoded controllers to generate plants able to switch gene expression selectively depending on oxygen availability, thereby providing a proof of concept for the potential of synthetic biology to assist agricultural practices in environments with variable oxygen provision.

Understanding the influence of disorder on the exciton dynamics and energy transfer in Zn-phthalocyanine H-aggregates.

The photophysics of 9(19),16(17),23(24)-tri-tert-butyl-2-[ethynyl-(4-carboxymethyl)phenyl]phthalocyaninatozinc(ii) and its H-aggregates is studied in different solvents by means of ultrafast non-linear optical spectroscopy and computational modeling. In non-coordinating solvents, both stationary and time-resolved spectroscopies highlight the formation of extended molecular aggregates, whose dimension and spectral properties depends on the concentration. In all the explored experimental conditions, time-resolved transient absorption experiments show multi exponential decay of the signals. Additional insights into the excited state relaxation mechanisms of the system is obtained with 2D electronic spectroscopy, which is employed to compare the deactivation channels in the absence or presence of aggregates. In ethanol and diethylether, where only monomers are present, an ultrafast relaxation process among the two non-degenerate Q-states of the molecule is evidenced by the appearance of a cross peak in the 2D-maps. In chloroform or CCl4, where disordered H-aggregates are formed, an energy transfer channel among aggregates with different composition and size is observed, leading to the non-radiative decay towards the lower energy dark state of the aggregates. Efficient coupling between less and more aggregated species is highlighted in two-dimensional electronic spectra by the appearance of a cross peak. The kinetics and intensity of the latter depend on the concentration of the solution. Finally, the linear spectroscopic properties of the aggregate are reproduced using a simplified structural model of an extended aggregate, based on Frenkel Hamiltonian Calculations and on an estimate of the electronic couplings between each dimer composing the aggregate computed at DFT level.

Coherence in carotenoid-to-chlorophyll energy transfer.

The subtle details of the mechanism of energy flow from carotenoids to chlorophylls in biological light-harvesting complexes are still not fully understood, especially in the ultrafast regime. Here we focus on the antenna complex peridinin-chlorophyll a-protein (PCP), known for its remarkable efficiency of excitation energy transfer from carotenoids-peridinins-to chlorophylls. PCP solutions are studied by means of 2D electronic spectroscopy in different experimental conditions. Together with a global kinetic analysis and multiscale quantum chemical calculations, these data allow us to comprehensively address the contribution of the potential pathways of energy flow in PCP. These data support dominant energy transfer from peridinin S2 to chlorophyll Qy state via an ultrafast coherent mechanism. The coherent superposition of the two states is functional to drive population to the final acceptor state, adding an important piece of information in the quest for connections between coherent phenomena and biological functions.

The role of charge-transfer states in the spectral tuning of antenna complexes of purple bacteria.

The LH2 antenna complexes of purple bacteria occur, depending on light conditions, in various different spectroscopic forms, with a similar structure but different absorption spectra. The differences are related to point changes in the primary amino acid sequence, but the molecular-level relationship between these changes and the resulting spectrum is still not well understood. We undertook a systematic quantum chemical analysis of all the main factors that contribute to the exciton structure, looking at how the environment modulates site energies and couplings in the B800-850 and B800-820 spectroscopic forms of LH2. A multiscale approach combining quantum chemistry and an atomistic classical embedding has been used where mutual polarization effects between the two parts are taken into account. We find that the loss of hydrogen bonds following amino acid changes can only explain a part of the observed blue-shift in the B850 band. The coupling of excitonic states to charge-transfer states, which is different in the two forms, contributes with a similar amount to the overall blue-shift.

The modeling of the absorption lineshape for embedded molecules through a polarizable QM/MM approach.

We present a computational strategy to simulate the absorption lineshape of a molecule embedded in a complex environment by using a polarizable QM/MM approach. This strategy is presented in two alternative formulations, one based on a molecular dynamics simulation of the structural fluctuations of the system and the other using normal modes and harmonic frequencies calculated on optimized geometries. The comparison for the case of a chromophore within a strongly inhomogeneous and structured environment, namely the intercalation pocket of DNA, shows that the MD-based approach is able to reproduce the experimental spectral bandshape. In contrast, the static approach overestimates the vibronic coupling, resulting in a much broader band.

Atomic Detail of Protein Folding Revealed by an Ab Initio Reappraisal of Circular Dichroism.

Circular dichroism (CD) is known to be an excellent tool for the determination of protein secondary structure due to fingerprint signatures of α and ß domains. However, CD spectra are also sensitive to the 3D arrangement of the chain as a result of the excitonic nature of additional signals due to the aromatic residues. This double sensitivity, when extended to time-resolved experiments, should allow protein folding to be monitored with high spatial resolution. To date, the exploitation of this very appealing idea has been limited, due to the difficulty in relating the observed spectral evolution to specific configurations of the chain. Here, we demonstrate that the combination of atomistic molecular dynamics simulations of the folding pathways with a quantum chemical evaluation of the excitonic spectra provides the missing key. This is exemplified for the folding of canine milk lysozyme protein.

Frenkel-exciton decomposition analysis of circular dichroism and circularly polarized luminescence for multichromophoric systems.

Recently, a method to calculate the absorption and circular dichroism (CD) spectra based on the exciton coupling has been developed. In this work, the method was utilized for the decomposition of the CD and circularly polarized luminescence (CPL) spectra of a multichromophoric system into chromophore contributions for recently developed through-space conjugated oligomers. The method which has been implemented using rotatory strength in the velocity form and therefore it is gauge-invariant, enables us to evaluate the contribution from each chromophoric unit and locally excited state to the CD and CPL spectra of the total system. The excitonic calculations suitably reproduce the full calculations of the system, as well as the experimental results. We demonstrate that the interactions between electric transition dipole moments of adjacent chromophoric units are crucial in the CD and CPL spectra of the multichromophoric systems, while the interactions between electric and magnetic transition dipole moments are not negligible. © 2018 Wiley Periodicals, Inc.

EXAT: EXcitonic analysis tool.

We introduce EXcitonic Analysis Tool (EXAT), a program able to compute optical spectra of large excitonic systems directly from the output of quantum mechanical calculations performed with the popular Gaussian 16 package. The software is able to combine in an excitonic scheme the single-chromophore properties and exciton couplings to simulate energies, coefficients, and excitonic spectra (UV-vis, CD, and LD). The effect of the environment can also be included using a Polarizable Continuum Model. EXAT also presents a simple graphical user interface, which shows on-screen both site and exciton properties. To show the potential of the method, we report two applications on a a chiral perturbed BODIPY system and DNA G-quadruplexes, respectively. The program is available online at http://molecolab.dcci.unipi.it/tools/. © 2017 Wiley Periodicals, Inc.

Polarizable QM/Classical Approaches for the Modeling of Solvation Effects on UV-Vis and Fluorescence Spectra: An Integrated Strategy.

Hybrid methods combining quantum chemistry and classical models are largely used to describe solvent effects in absorption and emission processes of solvated chromophores. Here we compare three different formulations of these hybrid approaches, using a continuum, an atomistic, or a mixed description of the solvent. In all cases mutual polarization effects between the quantum and the classical subsystems are taken into account. As a molecular probe, 3-hydroxyflavone has been selected due to its rich photophysics, which involves different tautomeric and anionic forms. We show that a clear assignment of the measured spectroscopic signals to each specific form can be achieved by combining the different solvation models into an integrated and cost-effective strategy. Previously proposed mechanisms for the excited-state proton transfer (ESIPT), specific solvent perturbation effects on ESIPT, and solvent-assisted anion formation are also validated in terms of short- and long-range solvation effects.

Exciton transport in the PE545 complex: insight from atomistic QM/MM-based quantum master equations and elastic network models.

In this paper, we work out a parameterization of environmental noise within the Haken-Strobl-Reinenker (HSR) model for the PE545 light-harvesting complex, based on atomic-level quantum mechanics/molecular mechanics (QM/MM) simulations. We use this approach to investigate the role of various auto- and cross-correlations in the HSR noise tensor, confirming that site-energy autocorrelations (pure dephasing) terms dominate the noise-induced exciton mobility enhancement, followed by site energy-coupling cross-correlations for specific triplets of pigments. Interestingly, several cross-correlations of the latter kind, together with coupling-coupling cross-correlations, display clear low-frequency signatures in their spectral densities in the 30-70 [Formula: see text] region. These slow components lie at the limits of validity of the HSR approach, which requires that environmental fluctuations be faster than typical exciton transfer time scales. We show that a simple coarse-grained elastic-network-model (ENM) analysis of the PE545 protein naturally spotlights collective normal modes in this frequency range that represent specific concerted motions of the subnetwork of cysteines covalenty linked to the pigments. This analysis strongly suggests that protein scaffolds in light-harvesting complexes are able to express specific collective, low-frequency normal modes providing a fold-rooted blueprint of exciton transport pathways. We speculate that ENM-based mixed quantum classical methods, such as Ehrenfest dynamics, might be promising tools to disentangle the fundamental designing principles of these dynamical processes in natural and artificial light-harvesting structures.

Fine control of chlorophyll-carotenoid interactions defines the functionality of light-harvesting proteins in plants.

Photosynthetic antenna proteins can be thought of as "programmed solvents", which bind pigments at specific mutual orientations, thus tuning the overall energetic landscape and ensuring highly efficient light-harvesting. While positioning of chlorophyll cofactors is well understood and rationalized by the principle of an "energy funnel", the carotenoids still pose many open questions. Particularly, their short excited state lifetime (<25 ps) renders them potential energy sinks able to compete with the reaction centers and drastically undermine light-harvesting efficiency. Exploration of the orientational phase-space revealed that the placement of central carotenoids minimizes their interaction with the nearest chlorophylls in the plant antenna complexes LHCII, CP26, CP29 and LHCI. At the same time we show that this interaction is highly sensitive to structural perturbations, which has a profound effect on the overall lifetime of the complex. This links the protein dynamics to the light-harvesting regulation in plants by the carotenoids.

A Quantum Chemical Interpretation of Two-Dimensional Electronic Spectroscopy of Light-Harvesting Complexes.

Nonlinear electronic spectroscopies represent one of the most powerful techniques to study complex multichromophoric architectures. For these systems, in fact, linear spectra are too congested to be used to disentangle the many coupled vibroelectronic processes that are activated. By using a 2D approach, instead, a clear picture can be achieved, but only when the recorded spectra are combined with a proper interpretative model. So far, this has been almost always achieved through parametrized exciton Hamiltonians that necessarily introduce biases and/or arbitrary assumptions. In this study, a first-principles approach is presented that combines accurate quantum chemical descriptions with state-of-the-art models for the environment through the use of atomistic and polarizable embeddings. Slow and fast bath dynamics, along with exciton transport between the pigments, are included. This approach is applied to the 2DES spectroscopy of the Light-Harvesting 2 (LH2) complex of purple bacteria. Simulations are extended over the entire visible-near-infrared spectral region to cover both carotenoid and bacteriochlorophyll signals. Our results provide an accurate description of excitonic properties and relaxation pathways, and give an unprecedented insight into the interpretation of the spectral signatures of the measured 2D signals.

An Ab Initio Description of the Excitonic Properties of LH2 and Their Temperature Dependence.

The spectroscopic properties of light-harvesting (LH) antennae in photosyntehtic organisms represent a fingerprint that is unique for each specific pigment-protein complex. Because of that, spectroscopic observations are generally combined with structural data from X-ray crystallography to obtain an indirect representation of the excitonic properties of the system. Here, an alternative strategy is presented which goes beyond this empirical approach and introduces an ab initio computational description of both structural and electronic properties and their dependence on the temperature. The strategy is applied to the peripheral light-harvesting antenna complex (LH2) present in purple bacteria. By comparing this model with the one based on the crystal structure, a detailed, molecular level explanation of the absorption and circular dichroism (CD) spectra and their temperature dependence is achieved. The agreement obtained with the experiments at both low and room temperature lays the groundwork for an atomistic understanding of the excitation dynamics in the LH2 system.

Simulation of Electronic Circular Dichroism of Nucleic Acids: From the Structure to the Spectrum.

We present a quantum mechanical (QM) simulation of the electronic circular dichroism (ECD) of nucleic acids (NAs). The simulation combines classical molecular dynamics, to obtain the structure and its temperature-dependent fluctuations, with a QM excitonic model to determine the ECD. The excitonic model takes into account environmental effects through a polarizable embedding and uses a refined approach to calculate the electronic couplings in terms of full transition densities. Three NAs with either similar conformations but different base sequences or similar base sequences but different conformations have been investigated and the results were compared with experimental observations; a good agreement was seen in all cases. A detailed analysis of the nature of the ECD bands in terms of their excitonic composition was also carried out. Finally, a comparison between the QM and the DeVoe models clearly revealed the importance of including fluctuations of the excitonic parameters and of accurately determining the electronic couplings. This study demonstrates the feasibility of the ab initio simulation of the ECD spectra of NAs, that is, without the need of experimental structural or electronic data.

Photoprotection and triplet energy transfer in higher plants: the role of electronic and nuclear fluctuations.

Photosynthetic organisms employ several photoprotection strategies to avoid damage due to the excess energy in high light conditions. Among these, quenching of triplet chlorophylls by neighboring carotenoids (Cars) is fundamental in preventing the formation of singlet oxygen. Cars are able to accept the triplets from chlorophylls by triplet energy transfer (TET). We have here studied TET rates in CP29, a minor light-harvesting complex (LHC) of the Photosystem II in plants. A fully atomistic strategy combining classical molecular dynamics of the LHC in its natural environment with a hybrid time-dependent density functional theory/polarizable MM description of the TET is used. We find that the structural fluctuations of the pigment-protein complex can largely enhance the transfer rates with respect to those predicted using the crystal structure, reducing the triplet quenching times in the subnanosecond scale. These findings add a new perspective for the interpretation of the photoprotection function and its relation with structural motions of the LHC.

A fast but accurate excitonic simulation of the electronic circular dichroism of nucleic acids: how can it be achieved?

We present and discuss a simple and fast computational approach to the calculation of electronic circular dichroism spectra of nucleic acids. It is based on a exciton model in which the couplings are obtained in terms of the full transition-charge distributions, as resulting from TDDFT methods applied on the individual nucleobases. We validated the method on two systems, a DNA G-quadruplex and a RNA ß-hairpin whose solution structures have been accurately determined by means of NMR. We have shown that the different characteristics of composition and structure of the two systems can lead to quite important differences in the dependence of the accuracy of the simulation on the excitonic parameters. The accurate reproduction of the CD spectra together with their interpretation in terms of the excitonic composition suggest that this method may lend itself as a general computational tool to both predict the spectra of hypothetic structures and define clear relationships between structural and ECD properties.

Achieving linear scaling in computational cost for a fully polarizable MM/continuum embedding.

In this paper, we present a new, efficient implementation of a fully polarizable QM/MM/continuum model based on an induced-dipoles polarizable force field and on the Conductor-like Screening Model as a polarizable continuum in combination with a self-consistent field QM method. The paper focuses on the implementation of the MM/continuum embedding, where the two polarizable methods are fully coupled to take into account their mutual polarization. With respect to previous implementations, we achieve for the first time a linear scaling with respect to both the computational cost and the memory requirements without limitations on the molecular cavity shape. This is achieved thanks to the use of the recently developed ddCOSMO model for the continuum and the Fast Multipole Method for the force field, together with an efficient iterative procedure. Therefore, it becomes possible to include in the classical layer as much as several tens of thousands of atoms with a limited computational effort.

The role of magnetic-electric coupling in exciton-coupled ECD spectra: the case of bis-phenanthrenes.

Apparent exceptions to the exciton chirality method may arise for chromophores undergoing transitions which are both electric- and magnetic-dipole allowed, for example bis-phenanthrenes. We present a computational approach to calculate exciton-coupled CD spectra based on a quantum-mechanical description of the excitonic parameters, which also includes the solvent effects.

Limits and potentials of quantum chemical methods in modelling photosynthetic antennae.

Advances in electronic spectroscopies with femtosecond time resolution have provided new information on the excitonic processes taking place during the energy conversion in natural photosynthetic antennae. This has promoted the development of new theoretical protocols aiming at accurately describing the properties and mechanisms of exciton formation and relaxation. In this perspective, we provide an overview of the quantum chemical based approaches, trying to underline both the potentials of the methods and their weaknesses. In particular three main aspects will be analysed, the quantum mechanical description of excitonic parameters (site energies and couplings), the incorporation of environmental effects on these parameters through hybrid quantum/classical approaches, and the modelling of the dynamical coupling among such parameters and the vibrations of the pigment-protein complex.

Towards an ab initio description of the optical spectra of light-harvesting antennae: application to the CP29 complex of photosystem II.

Light-harvesting pigment-protein complexes (PPC) represent the fundamental units through which the photosynthetic organisms absorb sunlight and funnel the energy to the reaction centre for carrying out the primary energy conversion reactions of photosynthesis. Here we apply a multiscale computational strategy to a specific PPC present in the photosystem II of plants and algae (CP29) to investigate in what detail should the environment effects due to protein and membrane/solvent be included for an accurate description of optical spectra. We find that a refinement of the crystal structure is needed before any meaningful quantum chemical calculations of pigment transition energies can be performed. For this purpose we apply classical molecular dynamics simulations of the PPC within its natural environment and we perform ab initio computations of the exciton Hamiltonian of the complex, including the environment either implicitly by the polarizable continuum model (PCM) or explicitly using the polarizable QM/MM methodology (MMPol). However, PCM essentially leads to an unspecific redshift of all transition energies, and MMPol is able to reveal site-specific changes in the optical properties of the pigments. Based on the latter and the excitonic couplings obtained within a polarizable QM/MM methodology, optical spectra are calculated, which are in good qualitative agreement with experimental data. A weakness of the approach is however found in the overestimation of the fluctuations of the excitonic parameters of the pigments along the MD trajectory. An explanation for such a finding in terms of the limits of the force fields commonly used for protein cofactors is presented and discussed.