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.

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.

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.

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.

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.

Origins of the pH-Responsive Photoluminescence of Peptide-Functionalized Au Nanoclusters.

Ultrasmall peptide-protected gold nanoclusters are a promising class of bioresponsive material exhibiting pH-sensitive photoluminescence. We present a theoretical insight into the effect peptide-ligand environment has on pH-responsive fluorescence, with the aim of enhancing the rational design of gold nanoclusters for bioapplications. Employing a hybrid quantum/classical computational methodology, we systematically calculate deprotonation free energies of N-terminal cysteine amine groups in proximity to the inherently fluorescent core of Au25(Peptide)18 nanoclusters. We find that subtle changes in hexapeptide sequence alter the electrostatic environment and significantly shift the conventional N-terminal amine pKa expected for amino acids free-in-solution. Our findings provide an insight into how the deprotonation equilibrium of N-terminal amine and side chain carboxyl groups cooperatively respond to solution pH changes, explaining the experimentally observed, yet elusive, pH-responsive fluorescence of peptide-functionalized Au25 clusters.

Photophysical and Computational Insights into Ag(I) Complexation of Porphyrinic Covalent Cages Equipped with Triazoles-Incorporating Linkers.

The photophysical characterization of four supramolecular complexes based on covalent cages 2H-S-2H, 2H-L-2H, Zn-S-2H, and Zn-L-2H, consisting in either two free-base porphyrins or one Zn(II) porphyrin and one free-base porphyrin connected by four flexible linkers of different lengths incorporating triazole binding sites, and their Ag(I) complexation are reported. The complexation processes have been followed by means of absorption and emission spectroscopies, and a comprehensive computational study explains the behavior of the free-base porphyrin-containing cages. Absorption and emission features have been interpreted on the bases of conformational changes, metalation processes, and modification of energy transfer efficiencies occurring in the different cases. In all cages, except 2H-L-2H, the coordination of four Ag(I) ions to the lateral triazole groups of the linkers leads to the enlargement of their cavity. Only for 2H-L-2H is a different behavior observed, where the process of silver metalation of the porphyrins' core prevails.

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.

Tuning Proton Transfer Thermodynamics in SARS-CoV-2 Main Protease: Implications for Catalysis and Inhibitor Design.

The catalytic reaction in SARS-CoV-2 main protease is activated by a proton transfer (PT) from Cys145 to His41. The same PT is likely also required for the covalent binding of some inhibitors. Here we use a multiscale computational approach to investigate the PT thermodynamics in the apo enzyme and in complex with two potent inhibitors, N3 and the α-ketoamide 13b. We show that with the inhibitors the free energy cost to reach the charge-separated state of the active-site dyad is lower, with N3 inducing the most significant reduction. We also show that a few key sites (including specific water molecules) significantly enhance or reduce the thermodynamic feasibility of the PT reaction, with selective desolvation of the active site playing a crucial role. The approach presented is a cost-effective procedure to identify the enzyme regions that control the activation of the catalytic reaction and is thus also useful to guide the design of inhibitors.

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.

Inhibitor binding influences the protonation states of histidines in SARS-CoV-2 main protease.

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an attractive target for antiviral therapeutics. Recently, many high-resolution apo and inhibitor-bound structures of Mpro, a cysteine protease, have been determined, facilitating structure-based drug design. Mpro plays a central role in the viral life cycle by catalyzing the cleavage of SARS-CoV-2 polyproteins. In addition to the catalytic dyad His41-Cys145, Mpro contains multiple histidines including His163, His164, and His172. The protonation states of these histidines and the catalytic nucleophile Cys145 have been debated in previous studies of SARS-CoV Mpro, but have yet to be investigated for SARS-CoV-2. In this work we have used molecular dynamics simulations to determine the structural stability of SARS-CoV-2 Mpro as a function of the protonation assignments for these residues. We simulated both the apo and inhibitor-bound enzyme and found that the conformational stability of the binding site, bound inhibitors, and the hydrogen bond networks of Mpro are highly sensitive to these assignments. Additionally, the two inhibitors studied, the peptidomimetic N3 and an α-ketoamide, display distinct His41/His164 protonation-state-dependent stabilities. While the apo and the N3-bound systems favored N δ (HD) and N ϵ (HE) protonation of His41 and His164, respectively, the α-ketoamide was not stably bound in this state. Our results illustrate the importance of using appropriate histidine protonation states to accurately model the structure and dynamics of SARS-CoV-2 Mpro in both the apo and inhibitor-bound states, a necessary prerequisite for drug-design efforts.

Tuning Proton Transfer Thermodynamics in SARS-Cov-2 Main Protease: Implications for Catalysis and Inhibitor Design.

In this comutational work a hybrid quantum mechanics/molecular mechanics approach, the MD-PMM approach, is used to investigate the proton transfer reaction the activates the catalytic activity of SARS-CoV-2 main protease. The proton transfer thermodynamics is investigated for the apo ensyme (i.e., without any bound substrate or inhibitor) and in the presence of a inhibitor, N3, which was previously shown to covalently bind SARS-CoV-2 main protease.

Inhibitor binding influences the protonation states of histidines in SARS-CoV-2 main protease.

The main protease (M pro ) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an attractive target for antiviral therapeutics. Recently, many high-resolution apo and inhibitor-bound structures of M pro , a cysteine protease, have been determined, facilitating structure-based drug design. M pro plays a central role in the viral life cycle by catalyzing the cleavage of SARS-CoV-2 polyproteins. In addition to the catalytic dyad His41-Cys145, M pro contains multiple histidines including His163, His164, and His172. The protonation states of these histidines and the catalytic nu-cleophile Cys145 have been debated in previous studies of SARS-CoV M pro , but have yet to be investigated for SARS-CoV-2. In this work we have used molecular dynamics simulations to determine the structural stability of SARS-CoV-2 M pro as a function of the protonation assignments for these residues. We simulated both the apo and inhibitor-bound enzyme and found that the conformational stability of the binding site, bound inhibitors, and the hydrogen bond networks of M pro are highly sensitive to these assignments. Additionally, the two inhibitors studied, the peptidomimetic N3 and an α -ketoamide, display distinct His41/His164 protonation-state-dependent stabilities. While the apo and the N3-bound systems favored N δ (HD) and N ϵ (HE) protonation of His41 and His164, respectively, the α -ketoamide was not stably bound in this state. Our results illustrate the importance of using appropriate histidine protonation states to accurately model the structure and dynamics of SARS-CoV-2 M pro in both the apo and inhibitor-bound states, a necessary prerequisite for drug-design efforts.

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.

Fully Atomistic Multiscale Approach for pKa Prediction.

The ionization state of titratable amino acids strongly affects proteins structure and functioning in a large number of biological processes. It is therefore essential to be able to characterize the pKa of ionizable groups inside proteins and to understand its microscopic determinants in order to gain insights into many functional properties of proteins. A big effort has been devoted to the development of theoretical approaches for the prediction of deprotonation free energies, yet the accurate theoretical/computational calculation of pKa values is recognized as a current challenge. A methodology based on a hybrid quantum/classical approach is here proposed for the computation of deprotonation free energies. The method is applied to calculate the pKa of formic acid, methylammonium, and methanethiol, providing results in good agreement with the corresponding experimental estimates. The pKa is also calculated for aspartic acid and lysine as single residues in solution and for three aspartic/glutamic acids inside a well-characterized protein: hen egg white lysozyme. While for small molecules the method is able to deal with multiple protonation states of all titratable groups, this becomes computationally very expensive for proteins. The calculated pKa values for the single amino acids (except for the zwitterionic aspartic acid) and inside the protein display a systematic shift with respect to the experimental values that suggests that the fine balance between hydrophobic and polar interactions might be not accurately reproduced by the usual classical force-fields, thus affecting the computation of deprotonation free energies. The calculated pKa shifts inside the protein are in good agreement with the corresponding experimental ones (within 1 pKa unit), well reproducing the pKa changes due to the protein environment even in the case of large pKa shifts.

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.

Hydration Shell of Antifreeze Proteins: Unveiling the Role of Non-Ice-Binding Surfaces.

Antifreeze proteins (AFPs) have the ability to inhibit ice growth by binding to ice nuclei. Their ice-binding mechanism is still unclear, yet the hydration layer is thought to play a fundamental role. Here, we use molecular dynamics simulations to characterize the hydration shell of two AFPs and two non-AFPs. The calculated shell thickness and density of the AFPs do not feature any relevant difference with respect to the non-AFPs. Moreover, the hydration shell density is always higher than the bulk density and, thus, no low-density, ice-like layer is detected at the ice-binding surface (IBS) of AFPs. Instead, we observe local water-density differences in AFPs between the IBS (lower density) and the non-IBS (higher density). The lower solvent density at the ice-binding site can pave the way to the protein binding to ice nuclei, while the higher solvent density at the non-ice-binding surfaces might provide protection against ice growth.