Tuning the low-temperature phase behavior of aqueous ionic liquids.

Water's anomalous behavior is often explained using a two-liquid model, where two types of water, high-density liquid (HDL) and low-density liquid (LDL), can be separated via a liquid-liquid phase transition (LLPT) at low temperature. Mixtures of water and the ionic liquid hydrazinium trifluoroacetate were suggested to also show an LLPT but with the advantage that there is no rapid ice crystallization hampering its observation. It remains controversial whether these solutions exhibit an LLPT or are instead associated with complex phase separation phenomena. We here show detailed low-temperature calorimetry and diffraction experiments on aqueous solutions containing hydrazinium trifluoroacetate and other similar ionic liquids, all at a solute mole fraction of x = 0.175. Hydrazinium trifluoroacetate, ammonium trifluoroacetate, ethylammonium trifluoroacetate and hydrazinium pentafluoropropionate all boast exothermic transitions unrelated to crystallization as well as remarkable structural changes upon cooling into the glassy state. We propose a model inspired by micelle formation and decomposition in surfactant solutions, which is complemented by MD simulations and allows rationalizing the rich phase behavior of our mixtures during cooling. The fundamental aspect of the model is the hydrophobic nature of fluorinated anions that enables aggregation, which is reversed upon cooling and culminates in the remarkable exothermic first-order transition observed at low temperature. That is, we assign the first-order transition not to an LLPT but to phase-separations similar to the ones when falling below the Krafft temperature. All other solutions merely show simple vitrification behavior. Still, they exhibit distinct differences in liquid fragility, which is decreased continuously with decreasing hydrophobicity of the anions. This might enable the systematic tuning of ionic liquids with the goal of designing aqueous solutions of specific fragility.

Early prediction of spinodal-like relaxation events in supercooled liquid water.

Several computational studies on different water models reported evidence of a phase transition in supercooled conditions between two liquid states of water differing in density: the high-density liquid (HDL) and the low-density liquid (LDL). Yet, conclusive experimental evidence of the existence of a phase transition between the two liquid water phases could not be obtained due to fast crystallization in the region where the phase transition should occur. For the same reason, the investigation of possible transition mechanisms between the two phases is committed to computational investigations. In this work, we simulate an out-of-equilibrium temperature-induced transition from the LDL to the HDL-like state in the TIP4P/2005 water model. To structurally characterize the system relaxation, we use the node total communicability (NTC) we recently proposed as an effective order parameter to discriminate the two liquid phases differing in density. We find that the relaxation process is compatible with a spinodal-like scenario. We observe the formation of HDL-like domains in the LDL phase and we characterize their fluctuating behavior and subsequent coarsening and stabilization. Furthermore, we find that the formation of stable HDL-like domains is favored in the regions where the early formation of small patches of highly connected HDL-like molecules (i.e., with very high NTC values) is observed. Besides characterizing the LDL- to HDL-like relaxation from a structural point of view, these results also show that the NTC order parameter can serve as an early-time predictor of the regions from which the transition process initiates.

Mechanism and Dynamics of Photodecarboxylation Catalyzed by Lactate Monooxygenase.

Photoenzymes are a rare class of biocatalysts that use light to facilitate chemical reactions. Many of these catalysts utilize a flavin cofactor to absorb light, suggesting that other flavoproteins might have latent photochemical functions. Lactate monooxygenase is a flavin-dependent oxidoreductase previously reported to mediate the photodecarboxylation of carboxylates to afford alkylated flavin adducts. While this reaction holds a potential synthetic value, the mechanism and synthetic utility of this process are unknown. Here, we combine femtosecond spectroscopy, site-directed mutagenesis, and a hybrid quantum-classical computational approach to reveal the active site photochemistry and the role the active site amino acid residues play in facilitating this decarboxylation. Light-induced electron transfer from histidine to flavin was revealed, which has not been reported in other proteins. These mechanistic insights enable the development of catalytic oxidative photodecarboxylation of mandelic acid to produce benzaldehyde, a previously unknown reaction for photoenzymes. Our findings suggest that a much wider range of enzymes have the potential for photoenzymatic catalysis than has been realized to date.

A statistical mechanical model of supercooled water based on minimal clusters of correlated molecules.

In this paper, we apply a theoretical model for fluid state thermodynamics to investigate simulated water in supercooled conditions. This model, which we recently proposed and applied to sub- and super-critical fluid water [Zanetti-Polzi et al., J. Chem. Phys. 156(4), 44506 (2022)], is based on a combination of the moment-generating functions of the enthalpy and volume fluctuations as provided by two gamma distributions and provides the free energy of the system as well as other relevant thermodynamic quantities. The application we make here provides a thermodynamic description of supercooled water fully consistent with that expected by crossing the liquid-liquid Widom line, indicating the presence of two distinct liquid states. In particular, the present model accurately reproduces the Widom line temperatures estimated with other two-state models and well describes the heat capacity anomalies. Differently from previous models, according to our description, a cluster of molecules that extends beyond the first hydration shell is necessary to discriminate between the statistical fluctuation regimes typical of the two liquid states.

A general statistical mechanical model for fluid system thermodynamics: Application to sub- and super-critical water.

We propose in this paper a theoretical model for fluid state thermodynamics based on modeling the fluctuation distributions and, hence, the corresponding moment generating functions providing the free energy of the system. Using the relatively simple and physically coherent gamma model for the fluctuation distributions, we obtain a complete theoretical equation of state, also giving insight into the statistical/molecular organization and phase or pseudo-phase transitions occurring under the sub- and super-critical conditions, respectively. Application to sub- and super-critical fluid water and a comparison with the experimental data show that this model provides an accurate description of fluid water thermodynamics, except close to the critical point region where limited but significant deviations from the experimental data occur. We obtain quantitative evidence of the correspondence between the sub- and super-critical thermodynamic behaviors, with the super-critical water pseudo-liquid and pseudo-gas phases being the evolution of the sub-critical water liquid and gas phases, respectively. Remarkably, according to our model, we find that for fluid water the minimal subsystem corresponding to either the liquid-like or the gas-like condition includes an infinite number of molecules in the sub-critical regime (providing the expected singularities due to macroscopic phase transitions) but only five molecules in the super-critical regime (coinciding with the minimal possible hydrogen-bonding cluster), thus suggesting that the super-critical regime be characterized by the coexistence of nanoscopic subsystems in either the pseudo-liquid or the pseudo-gas phase with each subsystem fluctuating between forming and disrupting the minimal hydrogen-bonding network.

Segregation on the nanoscale coupled to liquid water polyamorphism in supercooled aqueous ionic-liquid solution.

The most intriguing hypothesis explaining many water anomalies is a metastable liquid-liquid phase transition (LLPT) at high pressure and low temperatures, experimentally hidden by homogeneous nucleation. Recent infrared spectroscopic experiments showed that upon addition of hydrazinium trifluoroacetate to water, the supercooled ionic solution undergoes a sharp, reversible LLPT at ambient pressure, possible offspring of that in pure water. Here, we calculate the temperature-dependent signature of the OH-stretching band, reporting on the low/high density phase of water, in neat water and in the same experimentally investigated ionic solution. The comparison between the infrared signature of the pure liquid and that of the ionic solution can be achieved only computationally, providing insight into the nature of the experimentally observed phase transition and allowing us to investigate the effects of ionic compounds on the high to low density supercooled liquid water transition. We show that the experimentally observed crossover behavior in the ionic solution can be reproduced only if the phase transition between the low- and high-density liquid states of water is coupled to a mixing-unmixing transition between the water component and the ions: at low temperatures, water and ions are separated and the water component is a low density liquid. At high temperatures, water and ions get mixed and the water component is a high-density liquid. The separation at low temperatures into ion-rich and ion-poor regions allows unveiling the polyamorphic nature of liquid water, leading to a crossover behavior resembling that observed in supercooled neat water under high pressure.

A computational insight into the relationship between side chain IR line shapes and local environment in fibril-like structures.

Infrared spectroscopy is a widely used technique to characterize protein structures and protein mediated processes. While the amide I band provides information on proteins' secondary structure, amino acid side chains are used as infrared probes for the investigation of protein reactions and local properties. In this paper, we use a hybrid quantum mechanical/classical molecular dynamical approach based on the perturbed matrix method to compute the infrared band due to the C=O stretching mode of amide-containing side chains. We calculate, at first, the infrared band of zwitterionic glutamine in water and obtain results in very good agreement with the experimental data. Then, we compute the signal arising from glutamine side chains in a microcrystal of the yeast prion Sup35-derived peptide, GNNQQNY, with a fibrillar structure. The infrared bands obtained by selective isotopic labeling of the two glutamine residues, Q4 and Q5, of each peptide were experimentally used to investigate the local hydration in the fibrillar microcrystal. The experimental spectra of the two glutamine residues, which experience different hydration environments, feature different spectral signals that are well reproduced by the corresponding calculated spectra. In addition, the analysis of the simulated spectra clarifies the molecular origin of the experimentally observed spectroscopic differences that arise from the different local electric field experienced by the two glutamine residues, which is, in turn, determined by a different hydrogen bonding pattern.

Allosteric Control of Naphthalene Diimide Encapsulation and Electron Transfer in Porphyrin Containers: Photophysical Studies and Molecular Dynamics Simulation.

The complexation processes of N,N'-dibutyl-1,4,5,8-naphthalene diimide (NDI) into two types of π-electron-rich molecular containers consisting of two Zn(II)-porphyrins connected by four flexible linkers of two different lengths, were characterized by means of absorption and emission spectroscopies and molecular dynamics simulation. Notably, the addition of NDI leads to a strong quenching of the fluorescence of both cages only when they are in an open conformation suitable for guest encapsulation, a situation triggered by silver(I) ions binding to the lateral triazoles. Molecular dynamics simulations confirm the fast binding of NDI, likely assisted by NDI-silver(I) interactions. Upon NDI complexation, the two porphyrin macrocycles get closer, with an optimized face to face orientation, suggesting an induced-fit mechanism through π-π interactions with the NDI aromatic cycle. Ultrafast transient absorption experiments allowed to identify the process of quenching of the Zn-porphyrin fluorescence as an efficient photoinduced electron transfer reaction between the cage porphyrin and the included NDI guest. The process occurs on fast and ultrafast time scales in the two complexes (1.5 ps and ≤300 fs) leading to a short-lived charge separated state (charge recombination lifetimes in the order of 30-40 ps). The combined computational and experimental approach used here is able to furnish a reliable model of the NDI-cage complexation mechanism and of the corresponding electron transfer reaction, attesting the allosteric control of both processes by the silver(I) ions.

Cooperative protein-solvent tuning of proton transfer energetics: carbonic anhydrase as a case study.

We investigate the coupling between the proton transfer (PT) energetics and the protein-solvent dynamics using the intra-molecular PT in wild type (wt) human carbonic anhydrase II and its ten-fold faster mutant Y7F/N67Q as a test case. We calculate the energy variation upon PT, and from that we also calculate the PT reaction free energy, making use of a hybrid quantum mechanics/molecular dynamics approach. In agreement with the experimental data, we obtain that the reaction free energy is basically the same in the two systems. Yet, we show that the instantaneous PT energy is on average lower in the mutant possibly contributing to the faster PT rate. Analysis of the contribution to the PT energetics of the solvent and of each protein residue, also not in the vicinity of the active site, provides evidence for electrostatic tuning of the PT energy arising from the combined effect of the solvent and the protein environment. These findings open up a way to the more general task of the rational design of mutants with either enhanced or reduced PT rate.

Modeling amino-acid side chain infrared spectra: the case of carboxylic residues.

Infrared (IR) spectroscopy is commonly utilized for the investigation of protein structures and protein-mediated processes. While the amide I band provides information on protein secondary structures, amino acid side chains are used as IR probes for the investigation of protein reactions, such as proton pumping in rhodopsins. In this work, we calculate the IR spectra of the solvated aspartic acid, with both zwitterionic and protonated backbones, and of a capped form, i.e. mimicking the aspartic acid residue in proteins, by means of molecular dynamics (MD) simulations and the perturbed matrix method (PMM). This methodology has already proved its good modeling capabilities for the amide I mode and is here extended to the treatment of protein side chains. The computed side chain vibrational signal is in very good agreement with the experimental one, well reproducing both the peak frequency position and the bandwidth. In addition, the MD-PMM approach proposed here is able to reproduce the small frequency shift (5-10 cm-1) experimentally observed between the protonated and zwitterionic forms, showing that such a shift depends on the excitonic coupling between the modes localized on the side chain and on the backbone in the protonated form. The spectrum of the capped form, in which the amide I band is also calculated, agrees well with the corresponding experimental spectrum. The reliable calculation of the vibrational bands of carboxyl-containing side chains provides a useful tool for the interpretation of experimental spectra.

Tip-Enhanced Infrared Difference-Nanospectroscopy of the Proton Pump Activity of Bacteriorhodopsin in Single Purple Membrane Patches.

Photosensitive proteins embedded in the cell membrane (about 5 nm thickness) act as photoactivated proton pumps, ion gates, enzymes, or more generally, as initiators of stimuli for the cell activity. They are composed of a protein backbone and a covalently bound cofactor (e.g. the retinal chromophore in bacteriorhodopsin (BR), channelrhodopsin, and other opsins). The light-induced conformational changes of both the cofactor and the protein are at the basis of the physiological functions of photosensitive proteins. Despite the dramatic development of microscopy techniques, investigating conformational changes of proteins at the membrane monolayer level is still a big challenge. Techniques based on atomic force microscopy (AFM) can detect electric currents through protein monolayers and even molecular binding forces in single-protein molecules but not the conformational changes. For the latter, Fourier-transform infrared spectroscopy (FTIR) using difference-spectroscopy mode is typically employed, but it is performed on macroscopic liquid suspensions or thick films containing large amounts of purified photosensitive proteins. In this work, we develop AFM-assisted, tip-enhanced infrared difference-nanospectroscopy to investigate light-induced conformational changes of the bacteriorhodopsin mutant D96N in single submicrometric native purple membrane patches. We obtain a significant improvement compared with the signal-to-noise ratio of standard IR nanospectroscopy techniques by exploiting the field enhancement in the plasmonic nanogap that forms between a gold-coated AFM probe tip and an ultraflat gold surface, as further supported by electromagnetic and thermal simulations. IR difference-spectra in the 1450-1800 cm-1 range are recorded from individual patches as thin as 10 nm, with a diameter of less than 500 nm, well beyond the diffraction limit for FTIR microspectroscopy. We find clear spectroscopic evidence of a branching of the photocycle for BR molecules in direct contact with the gold surfaces, with equal amounts of proteins either following the standard proton-pump photocycle or being trapped in an intermediate state not directly contributing to light-induced proton transport. Our results are particularly relevant for BR-based optoelectronic and energy-harvesting devices, where BR molecular monolayers are put in contact with metal surfaces, and, more generally, for AFM-based IR spectroscopy studies of conformational changes of proteins embedded in intrinsically heterogeneous native cell membranes.

Tyrosine absorption spectroscopy: Backbone protonation effects on the side chain electronic properties.

The UV-vis spectrum of Tyrosine and its response to different backbone protonation states have been studied by applying the Perturbed Matrix Method (PMM) in conjunction with molecular dynamics (MD) simulations. Herein, we theoretically reproduce the UV-vis absorption spectrum of aqueous solution of Tyrosine in its zwitterionic, anionic and cationic forms, as well as of aqua-p-Cresol (i.e., the moiety that constitutes the side chain portion of Tyrosine). To achieve a better accuracy in the MD sampling, the Tyrosine Force Field (FF) parameters were derived de novo via quantum mechanical calculations. The UV-vis absorption spectra are computed considering the occurring electronic transitions in the vertical approximation for each of the chromophore configurations sampled by the classical MD simulations, thus including the effects of the chromophore semiclassical structural fluctuations. Finally, the explicit treatment of the perturbing effect of the embedding environment permits to fully model the inhomogeneous bandwidth of the electronic spectra. Comparison between our theoretical-computational results and experimental data shows that the used model captures the essential features of the spectroscopic process, thus allowing to perform further analysis on the strict relationship between the quantum properties of the chromophore and the different embedding environments. © 2018 Wiley Periodicals, Inc.

Extending the perturbed matrix method beyond the dipolar approximation: comparison of different levels of theory.

Some years ago we developed a theoretical-computational hybrid quantum/classical methodology, the Perturbed Matrix Method (PMM), to be used in conjunction with molecular dynamics simulations for the investigation of chemical processes in complex systems, that proved to be a valuable tool for the simulation of relevant experimental observables, e.g., spectroscopic signals, reduction potentials, kinetic constants. In typical PMM calculations the quantum sub-part of the system, the quantum centre, is embedded into an external perturbing field providing a perturbation operator explicitly calculated up to the dipolar terms. In this paper we further develop the PMM approach, beyond the dipolar terms in the perturbation operator expansion, by including explicitly the quadrupolar terms and/or by expanding the perturbation operator on each atom of the quantum centre. These different levels of the perturbation operator expansion, providing different levels of theory, have been tested by calculating three different spectroscopic observables: the spectral signal of liquid water and aqueous benzene due to the lowest energy electronic excitation and the infrared amide I band of aqueous trans-N-methylacetamide. All the systems tested show that, even though the previous PMM level of theory is already capable of reproducing the main features of the spectral signal, the higher levels of theory improve the quantitative reproduction of the spectral details.

Computational evidence support the hypothesis of neuroglobin also acting as an electron transfer species.

Neuroglobin (Ngb) is a recently identified hexa-coordinated globin, expressed in the nervous system of humans. Its physiological role is still debated: one hypothesis is that Ngb serves as an electron transfer (ET) species, possibly by reducing cytochrome c and preventing it to initiate the apoptotic cascade. Here, we use the perturbed matrix method (PMM), a mixed quantum mechanics/molecular dynamics approach, to investigate the redox thermodynamics of two neuroglobins, namely the human Ngb and GLB-6 from invertebrate Caenorhabditis elegans. In particular, we calculate the reduction potential of the two globins, resulting in an excellent agreement with the experimental values, and we predict the reorganization energies, λ, which have not been determined experimentally yet. The calculated λ values match well those reported for known ET proteins and thereby support a potential involvement in vivo of the two globins in ET processes.

A dynamical approach to non-adiabatic electron transfers at the bio-inorganic interface.

A methodology is proposed to investigate electron transfer reactions between redox-active biomolecular systems (e.g. a protein) and inorganic surfaces. The whole system is modelled at the atomistic level using classical molecular dynamics - making an extensive sampling of the system's configurations possible - and the energies associated with the redox-active complex reduction are calculated using a hybrid quantum/classical approach along the molecular dynamics trajectory. The non-adiabaticity is introduced a posteriori using a Monte Carlo approach based on the Landau-Zener theory extended to treat a metal surface. This approach thus allows us to investigate the role of the energy fluctuations, determined by the dynamical evolution of the system, as well as the role of non-adiabaticity in affecting the kinetic rate of the electron transfer reaction. Most notably, it allows us to investigate the two contributions separately, hence achieving a detailed picture of the mechanisms that determine the rate. The analysis of the system configurations also allows us to relate the estimated electronic coupling to the structural and dynamic properties of the system. As a test case, the methodology is here applied to study the electron transfer reaction between cytochrome c and a gold surface. The results obtained explain the different electron transfer rates experimentally measured for two different concentrations of proteins on the electrode surface.

Extending the essential dynamics analysis to investigate molecular properties: application to the redox potential of proteins.

Here, a methodology is proposed to investigate the collective fluctuation modes of an arbitrary set of observables, maximally contributing to the fluctuation of another functionally relevant observable. The methodology, based on the analysis of fully classical molecular dynamics (MD) simulations, exploits the essential dynamics (ED) method, originally developed to analyse the collective motions in proteins. We apply this methodology to identify the residues that are more relevant for determining the reduction potential (E(0)) of a redox-active protein. To this aim, the fluctuation modes of the single-residue electrostatic potentials mostly contributing to the fluctuations of the total electrostatic potential (the main determinant of E(0)) are investigated for wild-type azurin and two of its mutants with a higher E(0). By comparing the results here obtained with a previous study on the same systems [Zanetti-Polzi et al., Org. Biomol. Chem., 2015, 13, 11003] we show that the proposed methodology is able to identify the key sites that determine E(0). This information can be used for a general deeper understanding of the molecular mechanisms on the basis of the redox properties of the proteins under investigation, as well as for the rational design of mutants with a higher or lower E(0). From the results of the present analysis we propose a new azurin mutant that, according to our calculations, shows a further increase of E(0).

A few key residues determine the high redox potential shift in azurin mutants.

The wide range of variability of the reduction potential (E(0)) of blue-copper proteins has been the subject of a large number of studies in the past several years. In particular, a series of azurin mutants have been recently rationally designed tuning E(0) over a very broad range (700 mV) without significantly altering the redox-active site [Marshall et al., Nature, 2009, 462, 113]. This clearly suggests that interactions outside the primary coordination sphere are relevant to determine E(0) in cupredoxins. However, the molecular determinants of the redox potential variability are still undisclosed. Here, by means of atomistic molecular dynamics simulations and hybrid quantum/classical calculations, the mechanisms that determine the E(0) shift of two azurin mutants with high potential shifts are unravelled. The reduction potentials of native azurin and of the mutants are calculated obtaining results in good agreement with the experiments. The analysis of the simulations reveals that only a small number of residues (including non-mutated ones) are relevant in determining the experimentally observed E(0) variation via site-specific, but diverse, mechanisms. These findings open the path to the rational design of new azurin mutants with different E(0).

Surface packing determines the redox potential shift of cytochrome c adsorbed on gold.

Thermodynamic and dynamic properties of iso-1-cytochrome c covalently bound to a bare gold surface are here investigated by large scale atomistic simulations. The reduction potential of the protein for low and high surface concentrations is calculated showing a good agreement with experimental estimates. The origin of the dependence of the reduction potential on the surface concentration is investigated and is demonstrated to stem from the changing polarizability of the environment surrounding the protein, a mechanism reminiscent of crowding effects. Moreover, structural analyses are performed revealing relevant changes induced by the presence of the electrode on the dynamic properties of cytochrome c. In particular, one of the two cavities previously identified on the protein surface [Bortolotti et al. J. Am. Chem. Soc., 2012, 134, 13670], and that reversibly open in cytochrome c freely diffusing in solution, is found to be deformed when the protein is adsorbed on gold. This modification exemplifies a mechanism that potentially leads to changes in the protein properties by surface-induced modification of its dynamical behavior.

Thermodynamic Evolution of a Metamorphic Protein: A Theoretical-Computational Study of Human Lymphotactin.

Metamorphic, or fold-switching, proteins feature different folds that are physiologically relevant. The human chemokine XCL1 (or Lymphotactin) is a metamorphic protein that features two native states, an [Formula: see text] and an all[Formula: see text] fold, which have similar stability at physiological condition. Here, extended molecular dynamics (MD) simulations, principal component analysis of atomic fluctuations and thermodynamic modeling based on both the configurational volume and free energy landscape, are used to obtain a detailed characterization of the conformational thermodynamics of human Lymphotactin and of one of its ancestors (as was previously obtained by genetic reconstruction). Comparison of our computational results with the available experimental data show that the MD-based thermodynamics can explain the experimentally observed variation of the conformational equilibrium between the two proteins. In particular, our computational data provide an interpretation of the thermodynamic evolution in this protein, revealing the relevance of the configurational entropy and of the shape of the free energy landscape within the essential space (i.e., the space defined by the generalized internal coordinates providing the largest, typically non-Gaussian, structural fluctuations).

Evidence for a high pKa of an aspartic acid residue in the active site of CALB by a fully atomistic multiscale approach.

Candida antarctica Lipase B (CALB) is a paradigm for the family of lipases. At pH 7, the optimal pH for catalysis, the protonation state of an aspartic acid of the active site (Asp134) could not be conclusively assigned. In fact, the pKa estimate provided by a widely used computational tool, namely PropKa, that predicts pKa values of ionizable groups in proteins based on the crystallographic structure, is only slightly above 7 (pKa = 7.25). This, along with the lack of an experimental evaluation, makes the assignment of its protonation state at neutral pH challenging. Here, we calculate the pKa of Asp134 by means of a fully atomistic multiscale computational approach based on classical molecular dynamics (MD) simulation and the perturbed matrix method (PMM), namely the MD-PMM approach. MD-PMM is able to take into account the dynamics of the system and, at the same time, to treat the deprotonation step at the quantum level. The calculations provide a pKa value of 8.9 ± 1.1, hence suggesting that Asp134 in CALB should be protonated at neutral, and even at slightly basic, pH.Communicated by Ramaswamy H. Sarma.