Deprotonation of formic, acetic acids and bicarbonate ion in slit silica nanopores at infinite dilution and in the presence of electrolytes.

Dielectric effects and the coupled electrostatics between the nanoconfined and the internal/external aqueous media contribute to the observed deviations of chemistry within the nanoconfined environment when compared with unconfined systems. A systematic understanding has remained elusive, especially with respect to background salt concentration and boundary condition effects like the nanopore surface chemistry and the reference state used to calculate free energies. We utilize molecular dynamics simulations along with thermodynamic integration to determine the free energy difference associated with acid-base chemistry in 2 nm and 4 nm slit pores open to a bulk-like reservoir. pKa increases are predicted when confining acetic acid, formic acid, and bicarbonate in the slits at infinite dilution conditions. We find that confinement weakens the acids, and the modulation of outer pore surface dipole magnitudes can tune the pKa shift values, suggesting that purely "intrinsic" electrostatic effect on confinement may not exist. At sufficiently high salt concentrations, the dielectric/electrostatic effects on pKa values diminish due to charge screening effects. These discoveries enable future modifications of nanopore chemistries to achieve desirable properties for industrial applications.

Molecular Mechanism of Labelling Functional Cysteines by Heterocyclic Thiones.

Heterocyclic thiones have recently been identified as reversible covalent warheads, consistent with their mild electrophilic nature. Little is known so far about their mechanism of action in labelling nucleophilic sidechains, especially cysteines. The vast number of tractable cysteines promotes a wide range of target proteins to examine; however, our focus was put on functional cysteines. We chose the main protease of SARS-CoV-2 harboring Cys145 at the active site that is a structurally characterized and clinically validated target of covalent inhibitors. We screened an in-house, cysteine-targeting covalent inhibitor library which resulted in several covalent fragment hits with benzoxazole, benzothiazole and benzimidazole cores. Thione derivatives and Michael acceptors were selected for further investigations with the objective of exploring the mechanism of inhibition of the thiones and using the thoroughly characterized Michael acceptors for benchmarking our studies. Classical and hybrid quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations were carried out that revealed a new mechanism of covalent cysteine labelling by thione derivatives, which was supported by QM and free energy calculations and by a wide range of experimental results. Our study shows that the molecular recognition step plays a crucial role in the overall binding of both sets of molecules.

Accurate calculation of affinity changes to the close state of influenza A M2 transmembrane domain in response to subtle structural changes of adamantyl amines using free energy perturbation methods in different lipid bilayers.

Experimental binding free energies of 27 adamantyl amines against the influenza M2(22-46) WT tetramer, in its closed form at pH 8, were measured by ITC in DPC micelles. The measured Kd's range is ~44 while the antiviral potencies (IC50) range is ~750 with a good correlation between binding free energies computed with Kd and IC50 values (r = 0.76). We explored with MD simulations (ff19sb, CHARMM36m) the binding profile of complexes with strong, moderate and weak binders embedded in DMPC, DPPC, POPC or a viral mimetic membrane and using different experimental starting structures of M2. To predict accurately differences in binding free energy in response to subtle changes in the structure of the ligands, we performed 18 alchemical perturbative single topology FEP/MD NPT simulations (OPLS2005) using the BAR estimator (Desmond software) and 20 dual topology calculations TI/MD NVT simulations (ff19sb) using the MBAR estimator (Amber software) for adamantyl amines in complex with M2(22-46) WT in DMPC, DPPC, POPC. We observed that both methods with all lipids show a very good correlation between the experimental and calculated relative binding free energies (r = 0.77-0.87, mue = 0.36-0.92 kcal mol-1) with the highest performance achieved with TI/MBAR and lowest performance with FEP/BAR in DMPC bilayers. When antiviral potencies are used instead of the Kd values for computing the experimental binding free energies we obtained also good performance with both FEP/BAR (r = 0.83, mue = 0.75 kcal mol-1) and TI/MBAR (r = 0.69, mue = 0.77 kcal mol-1).

Assessing the Performance of Non-Equilibrium Thermodynamic Integration in Flavodoxin Redox Potential Estimation.

Flavodoxins are enzymes that contain the redox-active flavin mononucleotide (FMN) cofactor and play a crucial role in numerous biological processes, including energy conversion and electron transfer. Since the redox characteristics of flavodoxins are significantly impacted by the molecular environment of the FMN cofactor, the evaluation of the interplay between the redox properties of the flavin cofactor and its molecular surroundings in flavoproteins is a critical area of investigation for both fundamental research and technological advancements, as the electrochemical tuning of flavoproteins is necessary for optimal interaction with redox acceptor or donor molecules. In order to facilitate the rational design of biomolecular devices, it is imperative to have access to computational tools that can accurately predict the redox potential of both natural and artificial flavoproteins. In this study, we have investigated the feasibility of using non-equilibrium thermodynamic integration protocols to reliably predict the redox potential of flavodoxins. Using as a test set the wild-type flavodoxin from Clostridium Beijerinckii and eight experimentally characterized single-point mutants, we have computed their redox potential. Our results show that 75% (6 out of 8) of the calculated reaction free energies are within 1 kcal/mol of the experimental values, and none exceed an error of 2 kcal/mol, confirming that non-equilibrium thermodynamic integration is a trustworthy tool for the quantitative estimation of the redox potential of this biologically and technologically significant class of enzymes.

Free energy simulations to study mutational effect of a conserved residue, Trp24, on stability of human serum retinol-binding protein.

Human serum retinol-binding protein (RBP) is a plasma transport protein for vitamin A. RBP is a prime subclass of lipocalins, which bind nonpolar ligands within a ß-barrel. To understand the role of Trp 24, one of the highly conserved residues in RBP, free energy simulations have been carried out to understand the effects of the mutations from Trp at position 24 to Leu, Phe, and Tyr in the apo-RBP on its thermal stability. We examine various unfolded systems to study the dependence of the free energy differences on the denatured structure. Our calculated free energy difference values for the three mutations are in excellent agreement with the experimental values when the initial coordinates of the seven-residue peptide segments truncated from the crystal structure are used for the denatured systems. Our free energy change differences for the Trp→Leu, Trp→Phe, and Trp→Tyr mutations are 2.50 ± 0.69, 2.58 ± 0.50, and 2.49 ± 0.48 kcal/mol, respectively, when the native-like seven-residue peptides are used as models for the denatured systems. The main contributions to the free energy change differences for the Trp24→Leu and Trp24→Phe mutations are mainly from van der Waals and covalent interactions, respectively. Electrostatic, van der Waals and covalent terms equally contribute to the free energy change difference for the Trp24→Tyr mutation. The free energy simulation helps understand the detailed microscopic mechanism of the stability of the RBP mutants relative to the wild type and the role of the highly conserved residue, Trp24, of the human RBP.Communicated by Ramaswamy H. Sarma.

Computer simulation of molecular recognition in biomolecular system: from in silico screening to generalized ensembles.

Prediction of ligand-receptor complex structure is important in both the basic science and the industry such as drug discovery. We report various computation molecular docking methods: fundamental in silico (virtual) screening, ensemble docking, enhanced sampling (generalized ensemble) methods, and other methods to improve the accuracy of the complex structure. We explain not only the merits of these methods but also their limits of application and discuss some interaction terms which are not considered in the in silico methods. In silico screening and ensemble docking are useful when one focuses on obtaining the native complex structure (the most thermodynamically stable complex). Generalized ensemble method provides a free-energy landscape, which shows the distribution of the most stable complex structure and semi-stable ones in a conformational space. Also, barriers separating those stable structures are identified. A researcher should select one of the methods according to the research aim and depending on complexity of the molecular system to be studied.

The effect of Mg2+ on Ca2+ binding to cardiac troponin C in hypertrophic cardiomyopathy associated TNNC1 variants.

Cardiac troponin C (cTnC) is the critical Ca2+ -sensing component of the troponin complex. Binding of Ca2+ to cTnC triggers a cascade of conformational changes within the myofilament that culminate in force production. Hypertrophic cardiomyopathy (HCM)-associated TNNC1 variants generally induce a greater degree and duration of Ca2+ binding, which may underly the hypertrophic phenotype. Regulation of contraction has long been thought to occur exclusively through Ca2+ binding to site II of cTnC. However, work by several groups including ours suggest that Mg2+ , which is several orders of magnitude more abundant in the cell than Ca2+ , may compete for binding to the same cTnC regulatory site. We previously used isothermal titration calorimetry (ITC) to demonstrate that physiological concentrations of Mg2+ may decrease site II Ca2+ -binding in both N-terminal and full-length cTnC. Here, we explore the binding of Ca2+ and Mg2+ to cTnC harbouring a series of TNNC1 variants thought to be causal in HCM. ITC and thermodynamic integration (TI) simulations show that A8V, L29Q and A31S elevate the affinity for both Ca2+ and Mg2+ . Further, L48Q, Q50R and C84Y that are adjacent to the EF hand binding motif of site II have a more significant effect on affinity and the thermodynamics of the binding interaction. To the best of our knowledge, this work is the first to explore the role of Mg2+ in modifying the Ca2+ affinity of cTnC mutations linked to HCM. Our results indicate a physiologically significant role for cellular Mg2+ both at baseline and when elevated on modifying the Ca2+ binding properties of cTnC and the subsequent conformational changes which precede cardiac contraction.

Thermodynamic Formation Properties of Point Defects in Germanium Crystal.

Point defects are crucial in determining the quality of germanium crystals. A quantitative understanding of the thermodynamic formation properties of the point defects is necessary for the subsequent control of the defect formation during crystal growth. Here, molecular dynamics simulations were employed to investigate the formation energies, total formation free energies and formation entropies of the point defects in a germanium crystal. As far as we know, this is the first time that the total formation free energies of point defects in a germanium crystal have been reported in the literature. We found that the formation energies increased slightly with temperature. The formation free energies decreased significantly with an increase in temperature due to the increase in entropy. The estimated total formation free energies at the melting temperature are ~1.3 eV for self-interstitial and ~0.75 eV for vacancy, corresponding to a formation entropy of ~15 kB for both types of point defects.

Modulation of human transthyretin stability by the mutations at histidine 88 studied by free energy simulation.

Human transthyretin (TTR) is a homotetrameric plasma protein associated with a high percentage of ß-sheet, which forms amyloid fibrils and accumulates in tissues or extracellular matrix to cause amyloid diseases. Free energy simulations based on all-atom molecular dynamics simulations were carried out to analyze the effects of the His88 → Arg, Phe, and Tyr mutations on the stability of human TTR. The calculated free energy change differences (ΔΔG) caused by the His → Arg, Phe, and Tyr mutations at position 88 are 6.48 ± 0.45, -9.99 ± 0.54, and 2.66 ± 0.33 kcal/mol, respectively. These calculated free energy change differences between wild type and the mutants are in excellent agreement with prior experimental values. Our simulation results show that the wild type of the TTR is more stable than H88R and H88Y mutants, whereas it is less stable than the H88F mutant. The free energy component analysis shows that the primary contribution to the free energy change difference (ΔΔG) for the His → Arg mutation arises from electrostatic interaction; the ΔΔG for the His → Phe mutation is from van der Waals and electrostatic interactions and that for the His → Tyr mutation from covalent interaction. The simulation results show that the free energy calculation with thermodynamic integration is beneficial for understanding the detailed microscopic mechanism of protein stability. The implications of the results for understanding stabilizing and destabilizing effect of the mutation and the contribution to protein stability are discussed.

Classical and Machine Learning Methods for Protein - Ligand Binding Free Energy Estimation.

Binding free energy estimation of drug candidates to their biomolecular target is one of the best quantitative estimators in computer-aided drug discovery. Accurate binding free energy estimation is still a challengeable task even after decades of research, along with the complexity of the algorithm, time-consuming procedures, and reproducibility issues. In this review, we have discussed the advantages and disadvantages of diverse free energy methods like Thermodynamic Integration (TI), Bennett's Acceptance Ratio (BAR), Free Energy Perturbation (FEP), and alchemical methods. Moreover, we discussed the possible application of the machine learning method in proteinligand binding free energy estimation.

The phase diagrams of beryllium and magnesium oxide at megabar pressures.

We performab initiosimulations of beryllium (Be) and magnesium oxide (MgO) at megabar pressures and compare their structural and thermodynamic properties. We make a detailed comparison of our two recently derived phase diagrams of Be (Wuet al2021Phys. Rev.B104014103) and MgO (Soubiran and Militzer 2020Phys. Rev. Lett.125175701) using the thermodynamic integration technique, as they exhibit striking similarities regarding their shape. We explore whether the Lindemann criterion can explain the melting temperatures of these materials through the calculation of the Debye temperature at high pressure. From our free energy calculations, we find that the melting line of both materials is well represented by the Simon-Glazel fitTm(P) =T0(1 +P/a)1/c, whereT0= 1564 K,a= 15.8037 GPa andc= 2.4154 for Be, whileT0= 3010 K,a= 10.5797 GPa andc= 2.8683 for the MgO in the B1. For the B2 phase, we use the valuesa= 26.1163 GPa andc= 2.2426. Both materials exhibit negative Clapeyron slopes on the boundaries between the two solid phases that are strongly affected by anharmonic effects, which also influence the location of the solid-solid-liquid triple point. We find that the quasi-harmonic approximation underestimates the stability range of the low-pressure phases, namely hcp for Be and B1 for MgO. We also compute the phonon dispersion relations at low and high pressure for each of the phases of these materials, and also explore how the phonon density of states is modified by temperature. Finally, we derive secondary shock Hugoniot curves in addition to the principal Hugoniot curve for both materials, and study their offsets in pressure between solid and liquid branches.

Thermodynamic integration combined with molecular dynamic simulations to explore the cross-resistance mechanism of isoniazid and ethionamide.

Tuberculosis is an ancient disease of mankind, and its causative bacterium is Mycobacterium tuberculosis. Isoniazid is one of the most effective first-line antituberculosis drugs. As prodrugs, it and its derivative ethionamide act on enoyl-acyl carrier protein reductase (InhA) after being oxidized in bacteria, and kill the bacteria by inhibiting the formation of M. tuberculosis cell walls. However, the S94A mutation of InhA causes M. tuberculosis to develop cross-resistance to isoniazid and ethionamide. This work is dedicated to studying the cross-resistance mechanism of isoniazid and ethionamide through theoretical calculations. First, thermodynamic integral simulations are used to accurately calculate the relative binding energy of two drugs in the mutant and wild-type system. Furthermore, through classic molecular dynamic simulations and molecular mechanics generalized-Born surface area calculation, some key residues are identified and the binding affinity of isoniazid and ethionamide reduced by 9-13 kcal/mol due to S94A mutation. The hydrogen bond between Ala94 and isoniazid (ethionamide) disappeared and the energy contribution of Ala94 decreased after the mutation. In addition, the dynamic network analysis indicated that the mutation of Ser94 also indirectly affected the conformation of key residues such as Met147, Thr196, and Leu97, resulting in a reduction in the energy contribution of these residues. Finally, the binding conformation of isoniazid and ethionamide has also undergone major changes. The obtained results could provide valuable information for the future molecular design to overcome the drug resistance.

Tissue-Nonspecific Alkaline Phosphatase (TNAP) as the Enzyme Involved in the Degradation of Nucleotide Analogues in the Ligand Docking and Molecular Dynamics Approaches.

Tissue-nonspecific alkaline phosphatase (TNAP) is known to be involved in the degradation of extracellular ATP via the hydrolysis of pyrophosphate (PPi). We investigated, using three different computational methods, namely molecular docking, thermodynamic integration (TI) and conventional molecular dynamics (MD), whether TNAP may also be involved in the utilization of ß,γ-modified ATP analogues. For that, we analyzed the interaction of bisphosphonates with this enzyme and evaluated the obtained structures using in silico studies. Complexes formed between pyrophosphate, hypophosphate, imidodiphosphate, methylenediphosphonic acid monothiopyrophosphate, alendronate, pamidronate and zoledronate with TNAP were generated and analyzed based on ligand docking, molecular dynamics and thermodynamic integration. The obtained results indicate that all selected ligands show high affinity toward this enzyme. The forming complexes are stabilized through hydrogen bonds, electrostatic interactions and van der Waals forces. Short- and middle-term molecular dynamics simulations yielded very similar affinity results and confirmed the stability of the protein and its complexes. The results suggest that certain effectors may have a significant impact on the enzyme, changing its properties.

Molecular dynamics simulations of ortho-carborane nano-diamond storage within the nonpolar channel cavities of a right-handed coiled-coil tetrabrachion nanotube.

Molecular dynamics simulations have been performed on a complex in which clusters of boron in the form of molecules of the nanodiamond ortho-carborane ( C 2 B 10 H 12 ) have been inserted into the four large nonpolar cavities of a nanotube of the right-handed coiled-coil ( R H C C ) t e t r a b r a c h i o n . The techniques of multi-configurational thermodynamic integration, steered molecular dynamics and umbrella sampling have been combined to investigate the energetics of storage of ortho-carborane in the cavities and to map out the free energy landscape of the RHCC - t e t r a b r a c h i o n - o r t h o - c a r b o r a n e complex along the central channel and along directions transverse to the central channel. The purpose of the study was to explore potential pathways for the diffusion of ortho-carborane between the cavities and the solvent and to assess the stability of the complex as a possible drug delivery system for boron neutron capture therapy (BNCT). The investigation reveals a complex free energy landscape with a multitude of peaks and valleys, all of which can be related to specific architectural elements of the RHCC - n a n o t u b e , and the activation barriers for ortho-carborane capture and release support the requirements for rapid cargo uptake coupled with tight binding to the cavities.

Free energy simulations to understand the effect of Met → Ala mutations at positions 205, 206 and 213 on stability of human prion protein.

Prion diseases are a family of infectious amyloid diseases affecting human and animals. Prion propagation in transmissible spongiform encephalopathies is associated with the unfolding and conversion of normal cellular prion protein into its pathogenic scrapie form. Understanding the fundamentals of prion protein aggregation caused by mutations is crucial to unravel the pathology of prion diseases. To help understand the contributions of individual residues to the stability of the human prion protein, we have carried out free energy simulations based on atomistic molecular dynamics trajectories. We focus on Met → Ala mutations at positions 205, 206 and 213, which are mostly buried residues located on helix 3 of the protein. The simulations predicted that all three mutations destabilize the prion protein. Changes in unfolding free energy upon mutation, ∆∆G, are 3.10 ± 0.79, 2.00 ± 0.26 and 3.06 ± 0.66 kcal/mol for M205A, M206A and M213A, respectively, in excellent agreement with the corresponding experimental values of 3.09 ± 0.28, 1.50 ± 0.34 and 3.12 ± 0.27 kcal/mol [T. Hart et al. (2009) PNAS 106, 5651-5656]. Component analysis indicates that the major contributions to the loss of protein stability arise from van der Waals interactions for the M205A and M206A mutations, and from van der Waals and covalent energy terms for M213A. Interestingly, while free energy contributions from a majority of residues neighboring the mutation sites tend to stabilize the wild type, there are a few residues stabilizing the mutant side chains. Our results show that this approach to free energy calculation can be very useful for understanding the detailed mechanism of human prion protein stability.

Binding of calcium and magnesium to human cardiac troponin C.

Cardiac muscle thin filaments are composed of actin, tropomyosin, and troponin that change conformation in response to Ca2+ binding, triggering muscle contraction. Human cardiac troponin C (cTnC) is the Ca2+-sensing component of the thin filament. It contains structural sites (III/IV) that bind both Ca2+ and Mg2+ and a regulatory site (II) that has been thought to bind only Ca2+. Binding of Ca2+ at this site initiates a series of conformational changes that culminate in force production. However, the mechanisms that underpin the regulation of binding at site II remain unclear. Here, we have quantified the interaction between site II and Ca2+/Mg2+ through isothermal titration calorimetry and thermodynamic integration simulations. Direct and competitive binding titrations with WT N-terminal cTnC and full-length cTnC indicate that physiologically relevant concentrations of both Ca2+/Mg2+ interacted with the same locus. Moreover, the D67A/D73A N-terminal cTnC construct in which two coordinating residues within site II were removed was found to have significantly reduced affinity for both cations. In addition, 1 mM Mg2+ caused a 1.4-fold lower affinity for Ca2+. These experiments strongly suggest that cytosolic-free Mg2+ occupies a significant population of the available site II. Interaction of Mg2+ with site II of cTnC likely has important functional consequences for the heart both at baseline as well as in diseased states that decrease or increase the availability of Mg2+, such as secondary hyperparathyroidism or ischemia, respectively.

Well-tempered MCMC simulations for population pharmacokinetic models.

A full Bayesian statistical treatment of complex pharmacokinetic or pharmacodynamic models, in particular in a population context, gives access to powerful inference, including on model structure. Markov Chain Monte Carlo (MCMC) samplers are typically used to estimate the joint posterior parameter distribution of interest. Among MCMC samplers, the simulated tempering algorithm (TMCMC) has a number of advantages: it can sample from sharp multi-modal posteriors; it provides insight into identifiability issues useful for model simplification; it can be used to compute accurate Bayes factors for model choice; the simulated Markov chains mix quickly and have assured convergence in certain conditions. The main challenge when implementing this approach is to find an adequate scale of auxiliary inverse temperatures (perks) and associated scaling constants. We solved that problem by adaptive stochastic optimization and describe our implementation of TMCMC sampling in the GNU MCSim software. Once a grid of perks is obtained, it is easy to perform posterior-tempered MCMC sampling or likelihood-tempered MCMC (thermodynamic integration, which bridges the joint prior and the posterior parameter distributions, with assured convergence of a single sampling chain). We compare TMCMC to other samplers and demonstrate its efficient sampling of multi-modal posteriors and calculation of Bayes factors in two stylized case-studies and two realistic population pharmacokinetic inference problems, one of them involving a large PBPK model.

Thermodynamic Integration in 3n Dimensions without Biases or Alchemy for Protein Interactions.

Thermodynamic integration (TI), a powerful formalism for computing Gibbs free energy, has been implemented for many biophysical processes with alchemical schemes that require delicate human efforts to choose/design biasing potentials for sampling the desired biophysical events and to remove their artifactitious consequences afterwards. Theoretically, an alchemical scheme is exact but practically, an unsophisticated implementation of this exact formula can cause error amplifications. Small relative errors in the input parameters can be amplified many times in their propagation into the computed free energy [due to subtraction of similar numbers such as (105 ± 5)‒(100 ± 5) = 5 ± 7]. In this paper, we present an unsophisticated implementation of TI in 3n dimensions (3nD) (n=1,2,3…) for the potential of mean force along a 3nD path connecting one state in the bound state ensemble to one state in the unbound state ensemble. Fluctuations in these 3nD are integrated in the bound and unbound state ensembles but not along the 3nD path. Using TI3nD, we computed the standard binding free energies of three protein complexes: trometamol in Salmonella effector SpvD (n=1), biotin-avidin (n=2), and Colicin E9 endonuclease with cognate immunity protein Im9 (n=3). We employed three different protocols in three independent computations of E9-Im9 to show TI3nD's robustness. We also computed the hydration energies of ten biologically relevant compounds (n=1 for water, acetamide, urea, glycerol, trometamol, ammonium and n=2 for erythritol, 1,3-propanediol, xylitol, biotin). Each of the 15 computations is accomplishable within one (for hydration) to ten (for E9-Im9) days on an inexpensive GPU workstation. The computed results all agree with the available experimental data.

Performing solvation free energy calculations in LAMMPS using the decoupling approach.

The decoupling approach to solvation free energy calculations requires scaling the interactions between the solute and the solution with all intramolecular interactions preserved. This paper reports a new procedure that makes it possible to these calculations in LAMMPS. The procedure is tested against built-in GROMACS capabilities. The model compounds chosen to test our methodology are ethanol and biphenyl. The LAMMPS and GROMACS results obtained are in good agreement with each other. This work should help perform solvation free energy calculations in LAMMPS and/or other molecular dynamics software having no built-in functions to implement the decoupling approach.

Generating Excess Protons in Microsolvated Acid Clusters under Ambient Conditions: An Issue of Configurational Entropy versus Internal Energy.

Acid dissociation, and thus liberation of excess protons in small water droplets, impacts on diverse fields such as interstellar, atmospheric or environmental chemistry. At cryogenic temperatures below 1 K, it is now well established that as few as four water molecules suffice to dissociate the generic strong acid HCl, yet temperature-driven recombination sets in simply upon heating that cluster. Here, the fundamental question is posed of how many more water molecules are required to stabilize a hydrated excess proton at room temperature. Ab initio path integral simulations disclose that not five, but six water molecules are needed at 300 K to allow for HCl dissociation independently from nuclear quantum effects. In order to provide the molecular underpinnings of these observations, the classical and quantum free energy profiles were decomposed along the dissociation coordinate in terms of the corresponding internal energy and entropy profiles. What decides in the end about acid dissociation, and thus ion pair formation, in a specific microsolvated water cluster at room temperature is found to be a fierce competition between classical configurational entropy and internal energy, where the former stabilizes the undissociated state whereas the latter favors dissociation. It is expected that these are generic findings with broad implications on acid-base chemistry depending on temperature in small water assemblies.