Prevalence and mechanism of synergistic carboxylate-cation-water interactions in halophilic proteins.

The cytoplasmic proteins of some halophilic organisms remain stable and functional at multimolar concentrations of KCl, i.e., under conditions that most mesophilic proteins cannot withstand. Their stability arises from their unusual amino acid composition. The most dramatic difference between halophilic and mesophilic proteins is that the former are rich in acidic amino acids. It has been proposed that one of the evolutionary driving forces for this difference is the occurrence of synergistic interactions between multiple acidic amino acids at the surface of the protein, the potassium cations in solution, and water. We investigate this possibility with molecular dynamics simulations, using high-quality force fields for the protein-water, protein-ion, and ion-ion interactions. We create a rigorous thermodynamic definition of interactions between acidic amino acids on proteins that can be used to distinguish between synergistic, noninteracting and interfering interactions. Our results demonstrate that synergistic interactions between neighboring acidic amino acids in halophilic proteins are frequent at multimolar KCl concentration. Synergistic interactions have an electrostatic origin, and are associated with stronger water-to-carboxylate hydrogen bonds than for acidic amino acids without synergistic interactions. Synergistic interactions are not observed in minimal systems of carboxylates, indicating that the protein environment is critical for their emergence. Our results demonstrate that synergistic interactions are neither associated with rigid amino acid orientations nor with highly structured and slow moving water networks, as had been originally proposed. Moreover, synergistic interactions can also be found in unfolded protein conformations. However, because these conformations are only a small subset of the unfolded state ensemble, synergistic interactions should contribute to the net stabilization of the folded state.

Proteins maintain hydration at high [KCl] concentration regardless of content in acidic amino acids.

Proteins of halophilic organisms, which accumulate molar concentrations of KCl in their cytoplasm, have a much higher content in acidic amino acids than proteins of mesophilic organisms. It has been proposed that this excess is necessary to maintain proteins hydrated in an environment with low water activity, either via direct interactions between water and the carboxylate groups of acidic amino acids or via cooperative interactions between acidic amino acids and hydrated cations. Our simulation study of five halophilic proteins and five mesophilic counterparts does not support either possibility. The simulations use the AMBER ff14SB force field with newly optimized Lennard-Jones parameters for the interactions between carboxylate groups and potassium ions. We find that proteins with a larger fraction of acidic amino acids indeed have higher hydration levels, as measured by the concentration of water in their hydration shell and the number of water/protein hydrogen bonds. However, the hydration level of each protein is identical at low (bKCl = 0.15 mol/kg) and high (bKCl = 2 mol/kg) KCl concentrations; excess acidic amino acids are clearly not necessary to maintain proteins hydrated at high salt concentration. It has also been proposed that cooperative interactions between acidic amino acids in halophilic proteins and hydrated cations stabilize the folded protein structure and would lead to slower dynamics of the solvation shell. We find that the translational dynamics of the solvation shell is barely distinguishable between halophilic and mesophilic proteins; if such a cooperative effect exists, it does not have that entropic signature.

Unfolding mechanism and free energy landscape of single, stable, alpha helices at low pull speeds.

Single alpha helices (SAHs) stable in isolated form are often found in motor proteins where they bridge functional domains. Understanding the mechanical response of SAHs is thus critical to understand their function. The quasi-static force-extension relation of a small number of SAHs is known from single-molecule experiments. Unknown, or still controversial, are the molecular scale details behind those observations. We show that the deformation mechanism of SAHs pulled from the termini at pull speeds approaching the quasi-static limit differs from that of typical helices found in proteins, which are stable only when interacting with other protein domains. Using molecular dynamics simulations with atomistic resolution at low pull speeds previously inaccessible to simulation, we show that SAHs start unfolding from the termini at all pull speeds we investigated. Unfolding proceeds residue-by-residue and hydrogen bond breaking is not the main event determining the barrier to unfolding. We use the molecular simulation data to test the cooperative sticky chain model. This model yields excellent fits of the force-extension curves and quantifies the distance, xE = 0.13 nm, to the transition state, the natural frequency of bond vibration, ν0 = 0.82 ns-1, and the height, V0 = 2.9 kcal mol-1, of the free energy barrier associated with the deformation of single residues. Our results demonstrate that the sticky chain model could advantageously be used to analyze experimental force-extension curves of SAHs and other biopolymers.

Quantifying how step-wise fluorination tunes local solute hydrophobicity, hydration shell thermodynamics and the quantum mechanical contributions of solute-water interactions.

The ability to locally tune solute-water interactions and thus control the hydrophilic/hydrophobic character of a solute is key to control molecular self-assembly and to develop new drugs and biocatalysts; it has been a holy grail in synthetic chemistry and biology. To date, the connection between (i) the hydrophobicity of a functional group; (ii) the local structure and thermodynamics of its hydration shell; and (iii) the relative influence of van der Waals (dispersion) and electrostatic interactions on hydration remains unclear. We investigate this connection using spectroscopic, classical simulation and ab initio methods by following the transition from hydrophile to hydrophobe induced by the step-wise fluorination of methyl groups. Along the transition, we find that water-solute hydrogen bonds are progressively transformed into dangling hydroxy groups. Each structure has a distinct thermodynamic, spectroscopic and quantum-mechanical signature connected to the associated local solute hydrophobicity and correlating with the relative contribution of electrostatics and dispersion to the solute-water interactions.

Unexpected trends in the hydrophobicity of fluorinated amino acids reflect competing changes in polarity and conformation.

Fluorination can dramatically improve the thermal and proteolytic stability of proteins and their enzymatic activity. Key to the impact of fluorination on protein properties is the hydrophobicity of fluorinated amino acids. We use molecular dynamics simulations, together with a new fixed-charge, atomistic force field, to quantify the changes in hydration free energy, ΔGHyd, for amino acids with alkyl side chains and with 1 to 6 -CH → -CF side chain substitutions. Fluorination changes ΔGHyd by -1.5 to +2 kcal mol-1, but the number of fluorines is a poor predictor of hydrophobicity. Changes in ΔGHyd reflect two main contributions: (i) fluorination alters side chain-water interactions; we identify a crossover point from hydrophilic to hydrophobic fluoromethyl groups which may be used to estimate the hydrophobicity of fluorinated alkyl side-chains; (ii) fluorination alters the number of backbone-water hydrogen bonds via changes in the relative side chain-backbone conformation. Our results offer a road map to mechanistically understand how fluorination alters hydrophobicity of (bio)polymers.

Correction: Developing force fields when experimental data is sparse: AMBER/GAFF-compatible parameters for inorganic and alkyl oxoanions.

Correction for 'Developing force fields when experimental data is sparse: AMBER/GAFF-compatible parameters for inorganic and alkyl oxoanions' by Sadra Kashefolgheta et al., Phys. Chem. Chem. Phys., 2017, 19, 20593-20607.

Trimeric coiled coils expand the range of strength, toughness and dynamics of coiled coil motifs under shear.

Coiled coils are widespread protein motifs in nature, and promising building blocks for bio-inspired nanomaterials and nanoscale force sensors. Detailed structural insight into their mechanical response is required to understand their role in tissues and to design building blocks for applications. We use all-atom molecular dynamics simulations to elucidate the mechanical response of two types of coiled coils under shear: dimers and trimers. The amino acid sequences of both systems are similar, thus enabling universal (vs. system-specific) features to be identified. The trimer is mechanically more stable - it is both stronger and tougher - than the dimer, withstanding higher forces (127 pN vs. 49 pN at v = 10-3 nm ns-1) and dissipating up to five times more energy before rupture. The deformation mechanism of the trimer at all pull speeds is dominated by progressive helix unfolding. In contrast, at the lowest pull speeds, dimers deform by unfolding/refolding-assisted sliding. The additional helix in the trimer thus both determines the stability of the structure and affects the deformation mechanism, preventing helix sliding. The mechanical response of the coiled coils is not only sensitive to the oligomerization state but also to helix stability: preventing helix unfolding doubles the mechanical strength of the trimer, but decreases its toughness to half. Our results show that coiled coil trimers expand the range of coiled coil responses to an applied shear force. Altering the stability of individual helices against deformation emerges as one possible route towards fine-tuning this response, enabling the use of these motifs as nanomechanical building blocks.

Goodness of fit testing in dynamic single-molecule force spectroscopy.

Dynamic single-molecule force spectroscopy (SMFS) is a powerful method to characterize the mechanical stability of biomolecules. We address the problem that the standard manner of reporting the extracted energy landscape parameters does not reveal the intrinsic statistical errors associated with them. This problem becomes particularly relevant when SMFS is used to compare two or more different molecular systems. Here, we propose two methods that allow for a straightforward test of statistical significance. We illustrate the power of the methods by applying them to the experimental results obtained for three dimeric coiled coils of different lengths. Both methods are general and may be applied to any problem involving the fit of models with two correlated parameters.

Developing force fields when experimental data is sparse: AMBER/GAFF-compatible parameters for inorganic and alkyl oxoanions.

We present a set of Lennard-Jones parameters for classical, all-atom models of acetate and various alkylated and non-alkylated forms of sulfate, sulfonate and phosphate ions, optimized to reproduce their interactions with water and with the physiologically relevant sodium, ammonium and methylammonium cations. The parameters are internally consistent and are fully compatible with the Generalized Amber Force Field (GAFF), the AMBER force field for proteins, the accompanying TIP3P water model and the sodium model of Joung and Cheatham. The parameters were developed primarily relying on experimental information - hydration free energies and solution activity derivatives at 0.5 m concentration - with ab initio, gas phase calculations being used for the cases where experimental information is missing. The ab initio parameterization scheme presented here is distinct from other approaches because it explicitly connects gas phase binding energies to intermolecular interactions in solution. We demonstrate that the original GAFF/AMBER parameters often overestimate anion-cation interactions, leading to an excessive number of contact ion pairs in solutions of carboxylate ions, and to aggregation in solutions of divalent ions. GAFF/AMBER parameters lead to excessive numbers of salt bridges in proteins and of contact ion pairs between sodium and acidic protein groups, issues that are resolved by using the optimized parameters presented here.

Kinetics of formation of bile salt micelles from coarse-grained Langevin dynamics simulations.

We examine the mechanism of formation of micelles of dihydroxy bile salts using a coarse-grained, implicit solvent model and Langevin dynamics simulations. We find that bile salt micelles primarily form via addition and removal of monomers, similarly to surfactants with typical head-tail molecular structures, and not via a two-stage mechanism - involving formation of oligomers and their subsequent aggregation to form larger micelles - originally proposed for bile salts. The free energy barrier to removal of single bile monomers from micelles is ≈2kBT, much less than what has been observed for head-tail surfactants. Such a low barrier may be biologically relevant: it allows for rapid release of bile monomers into the intestine, possibly enabling the coverage of fat droplets by bile salt monomers and subsequent release of micelles containing fats and bile salts - a mechanism that is not possible for ionic head-tail surfactants of similar critical micellar concentrations.

Solvent-shared pairs of densely charged ions induce intense but short-range supra-additive slowdown of water rotation.

The question "Can ions exert supra-additive effects on water dynamics?" has had several opposing answers from both simulation and experiment. We address this ongoing controversy by investigating water reorientation in aqueous solutions of two salts with large (magnesium sulfate) and small (cesium chloride) effects on water dynamics using molecular dynamics simulations and classical, polarizable models. The salt models are reparameterized to reproduce properties of both dilute and concentrated solutions. We demonstrate that water rotation in concentrated MgSO4 solutions is unexpectedly slow, in agreement with experiment, and that the slowdown is supra-additive: the observed slowdown is larger than that predicted by assuming that the resultant of the extra forces induced by the ions on the rotating water molecules tilts the free energy landscape associated with water rotation. Supra-additive slow down is very intense but short-range, and is strongly ion-specific: in contrast to the long-range picture initially proposed based on experiment, we find that intense supra-additivity is limited to water molecules directly bridging two ions in solvent-shared ion pair configuration; in contrast to a non-ion-specific origin to supra-additive effects proposed from simulations, we find that the magnitude of supra-additive slowdown strongly depends on the identity of the cations and anions. Supra-additive slowdown of water dynamics requires long-lived solvent-shared ion pairs; long-lived ion pairs should be typical for salts of multivalent ions. We discuss the origin of the apparent disagreement between the various studies on this topic and show that the short-range cooperative slowdown scenario proposed here resolves the existing controversy.

Chain Sliding versus ß-Sheet Formation upon Shearing Single α-Helical Coiled Coils.

Coiled coils (CCs) are key building blocks of biogenic materials and determine their mechanical response to large deformations. Of particular interest is the observation that CC-based materials display a force-induced transition from α-helices to mechanically stronger ß-sheets (αßT). Steered molecular dynamics simulations predict that this αßT requires a minimum, pulling speed-dependent CC length. Here, de novo designed CCs with a length between four to seven heptads are utilized to probe if the transition found in natural CCs can be mimicked with synthetic sequences. Using single-molecule force spectroscopy and molecular dynamics simulations, these CCs are mechanically loaded in shear geometry and their rupture forces and structural responses to the applied load are determined. Simulations at the highest pulling speed (0.01 nm ns-1 ) show the appearance of ß-sheet structures for the five- and six-heptad CCs and a concomitant increase in mechanical strength. The αßT is less probable at a lower pulling speed of 0.001 nm ns-1 and is not observed in force spectroscopy experiments. For CCs loaded in shear geometry, the formation of ß-sheets competes with interchain sliding. ß-sheet formation is only possible in higher-order CC assemblies or in tensile-loading geometries where chain sliding and dissociation are prohibited.

Ultrafast reorientation of dangling OH groups at the air-water interface using femtosecond vibrational spectroscopy.

We report the real-time measurement of the ultrafast reorientational motion of water molecules at the water-air interface, using femtosecond time- and polarization-resolved vibrational sum-frequency spectroscopy. Vibrational excitation of dangling OH bonds along a specific polarization axis induces a transient anisotropy that decays due to the reorientation of vibrationally excited OH groups. The reorientation of interfacial water is shown to occur on subpicosecond time scales, several times faster than in the bulk, which can be attributed to the lower degree of hydrogen bond coordination at the interface. Molecular dynamics simulations of interfacial water dynamics are in quantitative agreement with experimental observations and show that, unlike in bulk, the interfacial reorientation occurs in a largely diffusive manner.

Adsorption of homopolypeptides on gold investigated using atomistic molecular dynamics.

We investigate the role of dynamics on adsorption of peptides to gold surfaces using all-atom molecular dynamics simulations in explicit solvent. We choose six homopolypeptides [Ala(10), Ser(10), Thr(10), Arg(10), Lys(10), and Gln(10)], for which experimental surface coverages are not correlated with amino acid level affinities for gold, with the idea that dynamic properties may also play a role. To assess dynamics we determine both conformational movement and flexibility of the peptide within a given conformation. Low conformational movement indicates stability of a given conformation and leads to less adsorption than homopolypeptides with faster conformational movement. Likewise, low flexibility within a given conformation also leads to less adsorption. Neither amino acid affinities nor dynamic considerations alone predict surface coverage; rather both quantities must be considered in peptide adsorption to gold surfaces.

Influence of Methylene Fluorination and Chain Length on the Hydration Shell Structure and Thermodynamics of Linear Diols.

The interplay between the local hydration shell structure, the length of hydrophobic solutes, and their identity (perfluorinated or not) remains poorly understood. We address this issue by combining Raman-multivariate curve resolution (Raman-MCR) spectroscopy, simulation, and quantum-mechanical calculations to quantify the thermodynamics and the first principle interactions behind the formation of defects in the hydration shell of alkyl-diol and perfluoroalkyl-diol chains. The hydration shell of the fluorinated diols contains substantially more defects than that of the nonfluorinated diols; these defects are water hydroxy groups that do not donate hydrogen bonds and which either point to the solute (radial-dangling OH) or not (nonradial-dangling OH). The number of radial-dangling OH defects per carbon decreases for longer chains and toward the interior of the fluorinated diols, mainly due to less favorable electrostatics and exchange interactions; nonradial-dangling OH defects per carbon increase with chain length. In contrast, the hydration shell of the nonfluorinated diols only contains radial-dangling defects, which become more abundant toward the center of the chain and for larger chains, predominantly because of more favorable dispersion interactions. These results have implications for how the folding of macromolecules, ligand binding to biomacromolecules, and chemical reactions at water-oil interfaces could be modified through the introduction of fluorinated groups or solvents.

Molecular mechanics of coiled coils loaded in the shear geometry.

Coiled coils are important nanomechanical building blocks in biological and biomimetic materials. A mechanistic molecular understanding of their structural response to mechanical load is essential for elucidating their role in tissues and for utilizing and tuning these building blocks in materials applications. Using a combination of single-molecule force spectroscopy (SMFS) and steered molecular dynamics (SMD) simulations, we have investigated the mechanics of synthetic heterodimeric coiled coils of different length (3-4 heptads) when loaded in shear geometry. Upon shearing, we observe an initial rise in the force, which is followed by a constant force plateau and ultimately strand separation. The force required for strand separation depends on the coiled coil length and the applied loading rate, suggesting that coiled coil shearing occurs out of equilibrium. This out-of-equilibrium behaviour is determined by a complex structural response which involves helix uncoiling, uncoiling-assisted sliding of the helices relative to each other in the direction of the applied force as well as uncoiling-assisted dissociation perpendicular to the force axis. These processes follow a hierarchy of timescales with helix uncoiling being faster than sliding and sliding being faster than dissociation. In SMFS experiments, strand separation is dominated by uncoiling-assisted dissociation and occurs at forces between 25-45 pN for the shortest 3-heptad coiled coil and between 35-50 pN for the longest 4-heptad coiled coil. These values are highly similar to the forces required for shearing apart short double-stranded DNA oligonucleotides, reinforcing the potential role of coiled coils as nanomechanical building blocks in applications where protein-based structures are desired.

Is the Solution Activity Derivative Sufficient to Parametrize Ion-Ion Interactions? Ions for TIP5P Water.

Biomolecular processes involve hydrated ions, and thus molecular simulations of such processes require accurate force-field parameters for these ions. In the best force-fields, both ion-water and anion-cation interactions are explicitly parametrized. First, the ion Lennard-Jones parameters are optimized to reproduce, for example, single ion solvation free energies; then ion-pair interactions are adjusted to match experimental activity or activity derivatives. Here, we apply this approach to derive optimized parameters for concentrated NaCl, KCl, MgCl2, and CaCl2 salt solutions, to be used with the TIP5P water model. These parameters are of interest because of a number of desirable properties of the TIP5P water model, especially for the simulation of carbohydrates. The results show, that this state of the art approach is insufficient, because the activity derivative often reaches a plateau near the target experimental value, for a wide range of parameter values. The plateau emerges from the interconversion between different types of ion pairs, so parameters leading to equally good agreement with the target solution activity or activity derivative yield very different solution structures. To resolve this indetermination, a second target property, such as the experimentally determined ion-ion coordination number, is required to uniquely determine anion-cation interactions. Simulations show that combining activity derivatives and coordination number as experimental target properties to parametrize ion-ion interactions, is a powerful method for reliable ion-water force field parametrization, and gives insight into the concentration of contact or solvent shared ion pairs in a wide range of salt concentrations. For the alkali and halide ions Li+, Rb+, Cs+, F-, Br-, and I-, we present ion-water parameters appropriate at infinite dilution only.

Cooperative slowdown of water rotation near densely charged ions is intense but short-ranged.

We investigate the reorientation dynamics of water at 300 K in solutions of magnesium sulfate and cesium chloride from classical atomistic molecular dynamics simulations using the "simple water model with four sites and negative Drude polarizability" (SWM4-NDP) and accompanying ion models; for SO4(2-), we derive SWM4-NDP-compatible parameters. Results indicate that pairs of ions have a cooperative effect on water rotation but do not support the model based on experiment whereby ion cooperativity increases the number of very slow water molecules well beyond the ions' first hydration shell. Instead, we find that cooperative slowdown beyond the first hydration shell is weak. Intense cooperative slowdown is limited to the first hydration shells, the magnitude of the slowdown being stronger for the multivalent ions. Cooperative effects for different salts differ in both the magnitude of rotational slowdown and the spatial range of the affected water subpopulations.

The free OD at the air/D2O interface is structurally and dynamically heterogeneous.

Air/water interfaces are both ubiquitous in the environment and technology and a useful model for hydrophobic solvation more generally. Previous experimental and computational studies have highlighted that molecular level markers of such an extended hydrophobic surface are broken hydrogen bonds and, as a result, OH groups that are not hydrogen bond donors: free OH. Understanding both the time-averaged structure and structural dynamics of these free OH thus plays a critical role in developing a quantitative, molecular level understanding of hydrophobic solvation. Here we show, by combining polarization-dependent vibrational sum frequency (VSF) spectroscopy and molecular dynamics simulation, that the free OD of D2O at the air/D2O interface is structurally and dynamically heterogeneous: that longer lived free OD groups tend to point closer to the surface normal, have a narrower orientational distribution, and are closer to the vapor phase. Knowledge of this structural heterogeneity should help link existing descriptions of hydrophobic solvation that focus either on the termination of the bulk hydrogen bond network or local density fluctuations. In addition the results of this study clarify that schemes to increase signal-to-noise ratios in VSF measurements by delaying the visible pulse relative to the infrared should be used only with independent constraints on the system's structural dynamics.