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Photosynthetic reaction centres harvest the energy content of sunlight by transporting electrons across an energy-transducing biological membrane. Here we use time-resolved serial femtosecond crystallography1 using an X-ray free-electron laser2 to observe light-induced structural changes in the photosynthetic reaction centre of Blastochloris viridis on a timescale of picoseconds. Structural perturbations first occur at the special pair of chlorophyll molecules of the photosynthetic reaction centre that are photo-oxidized by light. Electron transfer to the menaquinone acceptor on the opposite side of the membrane induces a movement of this cofactor together with lower amplitude protein rearrangements. These observations reveal how proteins use conformational dynamics to stabilize the charge-separation steps of electron-transfer reactions.
Assuntos
Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Bacterioclorofilas/metabolismo , Sítios de Ligação/efeitos dos fármacos , Clorofila/metabolismo , Clorofila/efeitos da radiação , Cristalografia , Citoplasma/metabolismo , Transporte de Elétrons/efeitos dos fármacos , Elétrons , Hyphomicrobiaceae/enzimologia , Hyphomicrobiaceae/metabolismo , Lasers , Modelos Moleculares , Oxirredução/efeitos da radiação , Feofitinas/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/efeitos da radiação , Prótons , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo , Vitamina K 2/metabolismoRESUMO
High-spin molecules allow for bottom-up qubit design and are promising platforms for magnetic sensing and quantum information science. Optical addressability of molecular electron spins has also been proposed in first-row transition-metal complexes via optically detected magnetic resonance (ODMR) mechanisms analogous to the diamond-nitrogen-vacancy color center. However, significantly less progress has been made on the front of metal-free molecules, which can deliver lower costs and milder environmental impacts. At present, most luminescent open-shell organic molecules are π-diradicals, but such systems often suffer from poor ground-state open-shell characters necessary to realize a stable ground-state molecular qubit. In this work, we use alternancy symmetry to selectively minimize radical-radical interactions in the ground state, generating π-systems with high diradical characters. We call them m-dimers, referencing the need to covalently link two benzylic radicals at their meta carbon atoms for the desired symmetry. Through a detailed electronic structure analysis, we find that the excited states of alternant hydrocarbon m-diradicals contain important symmetries that can be used to construct ODMR mechanisms leading to ground-state spin polarization. The molecular parameters are set in the context of a tris(2,4,6-trichlorophenyl)methyl (TTM) radical dimer covalently tethered at the meta position, demonstrating the feasibility of alternant m-diradicals as molecular color centers.
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Constant pH molecular dynamics (MD) is a powerful technique that allows the protonation state of residues to change dynamically, thereby enabling the study of pH dependence in a manner that has not been possible before. Recently, a constant pH implementation was incorporated into the GROMACS MD package. Although this implementation provides good accuracy and performance, manual modification and the preparation of simulation input files are required, which can be complicated, tedious, and prone to errors. To simplify and automate the setup process, we present phbuilder, a tool that automatically prepares constant pH MD simulations for GROMACS by modifying the input structure and topology as well as generating the necessary parameter files. phbuilder can prepare constant pH simulations from both initial structures and existing simulation systems, and it also provides functionality for performing titrations and single-site parametrizations of new titratable group types. The tool is freely available at www.gitlab.com/gromacs-constantph. We anticipate that phbuilder will make constant pH simulations easier to set up, thereby making them more accessible to the GROMACS user community.
Assuntos
Simulação de Dinâmica Molecular , Software , Concentração de Íons de HidrogênioRESUMO
The observation that materials can change their properties when placed inside or near an optical resonator has sparked a fervid interest in understanding the effects of strong light-matter coupling on molecular dynamics, and several approaches have been proposed to extend the methods of computational chemistry into this regime. Whereas the majority of these approaches have focused on modeling a single molecule coupled to a single cavity mode, changes to chemistry have so far only been observed experimentally when very many molecules are coupled collectively to multiple modes with short lifetimes. While atomistic simulations of many molecules coupled to multiple cavity modes have been performed with semi-classical molecular dynamics, an explicit description of cavity losses has so far been restricted to simulations in which only a very few molecular degrees of freedom were considered. Here, we have implemented an effective non-Hermitian Hamiltonian to explicitly treat cavity losses in large-scale semi-classical molecular dynamics simulations of organic polaritons and used it to perform both mean-field and surface hopping simulations of polariton relaxation, propagation, and energy transfer.
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Chromophore cis/trans photoisomerization is a fundamental process in chemistry and in the activation of many photosensitive proteins. A major task is understanding the effect of the protein environment on the efficiency and direction of this reaction compared to what is observed in the gas and solution phases. In this study, we set out to visualize the hula twist (HT) mechanism in a fluorescent protein, which is hypothesized to be the preferred mechanism in a spatially constrained binding pocket. We use a chlorine substituent to break the twofold symmetry of the embedded phenolic group of the chromophore and unambiguously identify the HT primary photoproduct. Through serial femtosecond crystallography, we then track the photoreaction from femtoseconds to the microsecond regime. We observe signals for the photoisomerization of the chromophore as early as 300 fs, obtaining the first experimental structural evidence of the HT mechanism in a protein on its femtosecond-to-picosecond timescale. We are then able to follow how chromophore isomerization and twisting lead to secondary structure rearrangements of the protein ß-barrel across the time window of our measurements.
Assuntos
Corantes , Proteínas , Cristalografia , Estrutura Secundária de ProteínaRESUMO
Aptamer selection against novel infections is a complicated and time-consuming approach. Synergy can be achieved by using computational methods together with experimental procedures. This study aims to develop a reliable methodology for a rational aptamer in silico et vitro design. The new approach combines multiple steps: (1) Molecular design, based on screening in a DNA aptamer library and directed mutagenesis to fit the protein tertiary structure; (2) 3D molecular modeling of the target; (3) Molecular docking of an aptamer with the protein; (4) Molecular dynamics (MD) simulations of the complexes; (5) Quantum-mechanical (QM) evaluation of the interactions between aptamer and target with further analysis; (6) Experimental verification at each cycle for structure and binding affinity by using small-angle X-ray scattering, cytometry, and fluorescence polarization. By using a new iterative design procedure, structure- and interaction-based drug design (SIBDD), a highly specific aptamer to the receptor-binding domain of the SARS-CoV-2 spike protein, was developed and validated. The SIBDD approach enhances speed of the high-affinity aptamers development from scratch, using a target protein structure. The method could be used to improve existing aptamers for stronger binding. This approach brings to an advanced level the development of novel affinity probes, functional nucleic acids. It offers a blueprint for the straightforward design of targeting molecules for new pathogen agents and emerging variants.
Assuntos
Aptâmeros de Nucleotídeos , COVID-19 , Aptâmeros de Nucleotídeos/química , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , SARS-CoV-2 , Técnica de Seleção de Aptâmeros , Glicoproteína da Espícula de CoronavírusRESUMO
Proton diffusion (PD) across biological membranes is a fundamental process in many biological systems, and much experimental and theoretical effort has been employed for deciphering it. Here, we report on a spectroscopic probe, which can be tightly tethered to the membrane, for following fast (nanosecond) proton transfer events on the surface of membranes. Our probe is composed of a photoacid that serves as our light-induced proton source for the initiation of the PD process. We use our probe to follow PD, and its pH dependence, on the surface of lipid vesicles composed of a zwitterionic headgroup, a negative headgroup, a headgroup that is composed only from the negative phosphate group, or a positive headgroup without the phosphate group. We reveal that the PD kinetic parameters are highly sensitive to the nature of the lipid headgroup, ranging from a fast lateral diffusion at some membranes to the escape of protons from surface to bulk (and vice versa) at others. By referring to existing theoretical models for membrane PD, we found that while some of our results confirm the quasi-equilibrium model, other results are in line with the nonequilibrium model.
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Coupling molecules to the confined light modes of an optical cavity is showing great promise for manipulating chemical reactions. However, to fully exploit this principle and use cavities as a new tool for controlling chemistry, a complete understanding of the effects of strong light-matter coupling on molecular dynamics and reactivity is required. While quantum chemistry can provide atomistic insight into the reactivity of uncoupled molecules, the possibilities to also explore strongly coupled systems are still rather limited due to the challenges associated with an accurate description of the cavity in such calculations. Despite recent progress in introducing strong coupling effects into quantum chemistry calculations, applications are mostly restricted to single or simplified molecules in ideal lossless cavities that support a single light mode only. However, even if commonly used planar mirror micro-cavities are characterized by a fundamental mode with a frequency determined by the distance between the mirrors, the cavity energy also depends on the wave vector of the incident light rays. To account for this dependency, called cavity dispersion, in atomistic simulations of molecules in optical cavities, we have extended our multi-scale molecular dynamics model for strongly coupled molecular ensembles to include multiple confined light modes. To validate the new model, we have performed simulations of up to 512 Rhodamine molecules in red-detuned Fabry-Pérot cavities. The results of our simulations suggest that after resonant excitation into the upper polariton at a fixed wave vector, or incidence angle, the coupled cavity-molecule system rapidly decays into dark states that lack dispersion. Slower relaxation from the dark state manifold into both the upper and lower bright polaritons causes observable photo-luminescence from the molecule-cavity system along the two polariton dispersion branches that ultimately evolves toward the bottom of the lower polariton branch, in line with experimental observations. We anticipate that the more realistic cavity description in our approach will help to better understand and predict how cavities can modify molecular properties.
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Photoactive yellow proteins (PYPs) make up a diverse class of blue-light-absorbing bacterial photoreceptors. Electronic excitation of the p-coumaric acid chromophore covalently bound within PYP results in triphasic quenching kinetics; however, the molecular basis of this behavior remains unresolved. Here we explore this question by examining the excitation-wavelength dependence of the photodynamics of the PYP from Halorhodospira halophila via a combined experimental and computational approach. The fluorescence quantum yield, steady-state fluorescence emission maximum, and cryotrapping spectra are demonstrated to depend on excitation wavelength. We also compare the femtosecond photodynamics in PYP at two excitation wavelengths (435 and 475 nm) with a dual-excitation-wavelength-interleaved pump-probe technique. Multicompartment global analysis of these data demonstrates that the excited-state photochemistry of PYP depends subtly, but convincingly, on excitation wavelength with similar kinetics with distinctly different spectral features, including a shifted ground-state beach and altered stimulated emission oscillator strengths and peak positions. Three models involving multiple excited states, vibrationally enhanced barrier crossing, and inhomogeneity are proposed to interpret the observed excitation-wavelength dependence of the data. Conformational heterogeneity was identified as the most probable model, which was supported with molecular mechanics simulations that identified two levels of inhomogeneity involving the orientation of the R52 residue and different hydrogen bonding networks with the p-coumaric acid chromophore. Quantum calculations were used to confirm that these inhomogeneities track to altered spectral properties consistent with the experimental results.
Assuntos
Proteínas de Bactérias/química , Halorhodospira halophila/química , Luz , Simulação de Dinâmica Molecular , Fotorreceptores Microbianos/química , Proteínas de Bactérias/genética , Halorhodospira halophila/genética , Ligação de Hidrogênio , Fotorreceptores Microbianos/genética , Relação Estrutura-AtividadeRESUMO
We describe a method to measure ultrafast protein structural changes using time-resolved wide-angle X-ray scattering at an X-ray free-electron laser. We demonstrated this approach using multiphoton excitation of the Blastochloris viridis photosynthetic reaction center, observing an ultrafast global conformational change that arises within picoseconds and precedes the propagation of heat through the protein. This provides direct structural evidence for a 'protein quake': the hypothesis that proteins rapidly dissipate energy through quake-like structural motions.
Assuntos
Transferência de Energia/efeitos da radiação , Lasers , Ficobiliproteínas/efeitos da radiação , Ficobiliproteínas/ultraestrutura , Espalhamento a Baixo Ângulo , Difração de Raios X/métodos , Ficobiliproteínas/química , Conformação Proteica/efeitos da radiação , Doses de RadiaçãoRESUMO
Biocompatible gold nanoclusters can be utilized as contrast agents in virus imaging. The labeling of viruses can be achieved noncovalently but site-specifically by linking the cluster to the hydrophobic pocket of a virus via a lipid-like pocket factor. We have estimated the binding affinities of three different pocket factors of echovirus 1 (EV1) in molecular dynamics simulations combined with non-equilibrium free-energy calculations. We have also studied the effects on binding affinities with a pocket factor linked to the Au102pMBA44 nanocluster in different protonation states. Although the absolute binding affinities are over-estimated for all the systems, the trend is in agreement with recent experiments.3 Our results suggest that the natural pocket factor (palmitic acid) can be replaced by molecules pleconaril (drug) and its derivative Kirtan1 that have higher estimated binding affinities. Our results also suggest that including the gold nanocluster does not decrease the affinity of the pocket factor to the virus, but the affinity is sensitive to the protonation state of the nanocluster, i.e., to pH conditions. The methodology introduced in this work helps in the design of optimal strategies for gold-virus bioconjugation for virus detection and manipulation.
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Capsídeo/metabolismo , Enterovirus/metabolismo , Ouro/química , Nanopartículas Metálicas/química , Antivirais/metabolismo , Sítios de Ligação , Capsídeo/química , Enterovirus/química , Ouro/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Simulação de Dinâmica Molecular , Oxidiazóis/metabolismo , Oxazóis , Ácido Palmítico/metabolismo , TermodinâmicaRESUMO
There has been considerable debate on the existence of a low-barrier hydrogen bond (LBHB) in the photoactive yellow protein (PYP). The debate was initially triggered by the neutron diffraction study of Yamaguchi et al. ( Proc. Natl. Acad. Sci., U. S. A. , 2009 , 106 , 440 - 444 ) who suggested a model in which a neutral Arg52 residue triggers the formation of the LBHB in PYP. Here, we present an alternative model that is consistent within the error margins of the Yamaguchi structure factors. The model explains an increased hydrogen bond length without nuclear quantum effects and for a protonated Arg52. We tested both models by calculations under crystal, solution, and vacuum conditions. Contrary to the common assumption in the field, we found that a single PYP in vacuum does not provide an accurate description of the crystal conditions but instead introduces strong artifacts, which favor a LBHB and a large 1H NMR chemical shift. Our model of the crystal environment was found to stabilize the two Arg52 hydrogen bonds and crystal water positions for the protonated Arg52 residue in free MD simulations and predicted an Arg52 pKa upshift with respect to PYP in solution. The crystal and solution environments resulted in almost identical 1H chemical shifts that agree with NMR solution data. We also calculated the effect of the Arg52 protonation state on the LBHB in 3D nuclear equilibrium density calculations. Only the charged crystal structure in vacuum supports a LBHB if Arg52 is neutral in PYP at the previously reported level of theory ( J. Am. Chem. Soc. , 2014 , 136 , 3542 - 3552 ). We attribute the anomalies in the interpretation of the neutron data to a shift of the potential minimum, which does not involve nuclear quantum effects and is transferable beyond the Yamaguchi structure.
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Artefatos , Proteínas de Bactérias/química , Fotorreceptores Microbianos/química , Vácuo , Cristalografia por Raios X , Ligação de Hidrogênio , Modelos Moleculares , Conformação Proteica , SoluçõesRESUMO
Reversibly switchable fluorescent proteins (RSFPs) are essential for high-resolution microscopy of biological samples, but the reason why these proteins are photochromic is still poorly understood. To address this problem, we performed molecular dynamics simulations of the fast switching Met159Thr mutant of the RSFP Dronpa. Our simulations revealed a ground state structural heterogeneity in the chromophore pocket that consists of three populations with one, two, or three hydrogen bonds to the phenolate moiety of the chromophore. By means of non-adiabatic quantum mechanics/molecular dynamics simulations, we demonstrated that the subpopulation with a single hydrogen bond is responsible for off-switching through photo-isomerization of the chromophore, whereas two or more hydrogen bonds inhibit the isomerization and promote fluorescence instead. While rational design of new RSFPs has so far focused on structure alone, our results suggest that structural heterogeneity must be considered as well.
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Proteínas Luminescentes/química , Processos Fotoquímicos , Ligação de Hidrogênio , Isomerismo , Simulação de Dinâmica Molecular , Teoria QuânticaRESUMO
The fluorescent protein Dronpa undergoes reversible photoswitching reactions between the bright "on" and dark "off" states via photoisomerization and proton transfer reactions. We report the room temperature crystal structure of the fast switching Met159Thr mutant of Dronpa at 2.0-Å resolution in the bright on state. Structural differences with the wild type include shifted backbone positions of strand ß8 containing Thr159 as well as an altered A-C dimer interface involving strands ß7, ß8, ß10, and ß11. The Met159Thr mutation increases the cavity volume for the p-hydroxybenzylidene-imidazolinone chromophore as a result of both the side chain difference and the backbone positional differences.
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Proteínas Luminescentes/química , Proteínas Luminescentes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Animais , Antozoários/genética , Cristalografia por Raios X , Proteínas Luminescentes/genética , Simulação de Dinâmica Molecular , Mutação , Proteínas Recombinantes/genética , TemperaturaRESUMO
The approximate density-functional tight-binding theory method DFTB3 has been implemented in the quantum mechanics/molecular mechanics (QM/MM) framework of the Gromacs molecular simulation package. We show that the efficient smooth particle-mesh Ewald implementation of Gromacs extends to the calculation of QM/MM electrostatic interactions. Further, we make use of the various free-energy functionalities provided by Gromacs and the PLUMED plugin. We exploit the versatility and performance of the current framework in three typical applications of QM/MM methods to solve biophysical problems: (i) ultrafast proton transfer in malonaldehyde, (ii) conformation of the alanine dipeptide, and (iii) electron-induced repair of a DNA lesion. Also discussed is the further development of the framework, regarding mostly the options for parallelization.
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Simulação por Computador , Teoria Quântica , Software , Ligação Proteica , TermodinâmicaRESUMO
The cellular energy machinery depends on the presence and properties of protons at or in the vicinity of lipid membranes. To asses the energetics and mobility of a proton near a membrane, we simulated an excess proton near a solvated DMPC bilayer at 323 K, using a recently developed method to include the Grotthuss proton shuttling mechanism in classical molecular dynamics simulations. We obtained a proton surface affinity of -13.0 ± 0.5 kJ mol(-1). The proton interacted strongly with both lipid headgroup and linker carbonyl oxygens. Furthermore, the surface diffusion of the proton was anomalous, with a subdiffusive regime over the first few nanoseconds, followed by a superdiffusive regime. The time- and distance dependence of the proton surface diffusion coefficient within these regimes may also resolve discrepancies between previously reported diffusion coefficients. Our simulations show that the proton anomalous surface diffusion originates from restricted diffusion in two different surface-bound states, interrupted by the occasional bulk-mediated long-range surface diffusion. Although only a DMPC membrane was considered in this work, we speculate that the restrictive character of the on-surface diffusion is highly sensitive to the specific membrane conditions, which can alter the relative contributions of the surface and bulk pathways to the overall diffusion process. Finally, we discuss the implications of our findings for the energy machinery.
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Bicamadas Lipídicas/química , Prótons , Difusão , Dimiristoilfosfatidilcolina/químicaRESUMO
We present Hydrogen Dynamics (HYDYN), a method that allows explicit proton transfer in classical force field molecular dynamics simulations at thermodynamic equilibrium. HYDYN reproduces the characteristic properties of the excess proton in water, from the special pair dance, to the continuous fluctuation between the limiting Eigen and Zundel complexes, and the water reorientation beyond the first solvation layer. Advantages of HYDYN with respect to existing methods are computational efficiency, microscopic reversibility, and easy parameterization for any force field.
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Simulação de Dinâmica Molecular , Prótons , Termodinâmica , Água/químicaRESUMO
In this work we present the vibrationally resolved optical absorption spectrum of p-hydroxybenzylidene-2,3-dimethylimidazolinone (HBDI), the green fluorescent protein (GFP) chromophore, computed at several levels of theory, including time-dependent DFT with various functionals and basis sets, CASSCF, CASPT2 and XMCQDPT2. We also investigated what happens to the spectrum if the ground- and excited-state geometries are optimized at different levels of theory (mixed approach), as has been used previously. The vibrationally resolved absorption spectra obtained by DFT, CASPT2 and XMCQDPT2 are very similar and consist of a main absorption peak and a shoulder that is â¼1500 cm(-1) higher in energy. The vibrational progression increases moderately with temperature. These spectra are in qualitative agreement with experimental action spectra, but much narrower and lack the long tail in the blue, even at high temperatures. Because our calculated emission spectra, which are equally narrow, are in good agreement with the emission of green fluorescent protein at 253 K, we argue that the action spectrum are too broad to be considered as the absorption spectrum. The CASSCF method and the mixed approaches overestimate the vibrational progressions with respect to CAM-B3LYP, CASPT2 and XMCQDPT2, due to inaccuracies in the geometric S0 âS1 displacements. Finally, we computed the vibronic spectra of four chromophore analogues with different substitutions on the rings and found that these substitutions hardly affect the lineshape in vacuum.
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Gases/química , Proteínas de Fluorescência Verde/química , Compostos de Benzil/química , Elétrons , Proteínas de Fluorescência Verde/metabolismo , Imidazolinas/química , Teoria Quântica , Espectrofotometria Infravermelho , Temperatura , TermodinâmicaRESUMO
The vibronic spectra of the green fluorescent protein chromophore analogues p-hydroxybenzylidene-2,3-dimethylimidazolinone (HBDI) and 3,5-tert-butyl-HBDI (35Bu) are similar in the vacuum, but very different in water or ethanol. To understand this difference, we have computed the vibrationally resolved solution spectra of these chromophores, using the polarizable continuum model (PCM) to account for solvent effects on the (harmonic) potential energy surfaces (PES). In agreement with experiment, we found that the vibrational progression increases with the polarity of the solvent, but we could neither reproduce the broadening, nor the large difference between the absorption spectra of HBDI and 35Bu. To account for the inhomogeneous broadening of the solution spectra, we used two approaches. In the first, we estimated the polar broadening from the solvent reorganization energy upon photo-excitation, using the state-specific PCM implementation. In the second, we estimated the broadening from the variance of the vertical excitation energies in molecular dynamics trajectories. Although we found good agreement for the lineshape of 35Bu in ethanol, and to a lesser extent in water, we highly underestimated the broadening for HBDI. To resolve this discrepancy, we explored the PES of HBDI in water and found that in contrast to the PCM result, the ground-state geometry is not planar in explicit solvent. We furthermore found that nonplanar geometries enhance the intramolecular charge transfer upon excitation. Therefore, the solvent reorganization and broadening are much larger and we speculate that the much broader spectrum of HBDI in water is due to the population of nonplanar geometries.
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Proteínas de Fluorescência Verde/química , Soluções/química , Compostos de Benzil/química , Elétrons , Ligação de Hidrogênio , Imidazolinas/química , Simulação de Dinâmica Molecular , Teoria Quântica , Solventes/química , Espectrofotometria Infravermelho , Temperatura , TermodinâmicaRESUMO
Molecular dynamics (MD) simulations are widely applied to estimate absolute binding free energies of protein-ligand and protein-protein complexes. A routinely used method for binding free energy calculations with MD is umbrella sampling (US), which calculates the potential of mean force (PMF) along a single reaction coordinate. Surprisingly, in spite of its widespread use, few validation studies have focused on the convergence of the free energy computed along a single path for specific cases, not addressing the reproducibility of such calculations in general. In this work, we therefore investigate the reproducibility and convergence of US along a standard distance-based reaction coordinate for various protein-protein and protein-ligand complexes, following commonly used guidelines for the setup. We show that repeating the complete US workflow can lead to differences of 2-20 kcal/mol in computed binding free energies. We attribute those discrepancies to small differences in the binding pathways. While these differences are unavoidable in the established US protocol, the popularity of the latter could hint at a lack of awareness of such reproducibility problems. To test if the convergence of PMF profiles can be improved if multiple pathways are sampled simultaneously, we performed additional simulations with an adaptive-biasing method, here the accelerated weight histogram (AWH) approach. Indeed, the PMFs obtained from AHW simulations are consistent and reproducible for the systems tested. To the best of our knowledge, our work is the first to attempt a systematic assessment of the pitfalls in one the most widely used protocols for computing binding affinities. We anticipate therefore that our results will provide an incentive for a critical reassessment of the validity of PMFs computed with US, and make a strong case to further benchmark the performance of adaptive-biasing methods for computing binding affinities.