Tuesday, January 17, 2017

Hydration water in drug design

Electrostatic interactions with lipid heads groups retard water molecules near the surface of a membrane. But how are those dynamics affected by a membrane protein? Lars Schäfer at the Ruhr University of Bochum and colleagues attempt to answer that question using (ODNP-enhanced) NMR and simulations to deduce water motions (O. Fisette et al., JACS 138, 11526; 2016 – paper here). They conclude that the water-protein interactions have a weaker retarding effect, and dominate only at distances of more than 10 Å above the membrane surface. Moreover, the protein (here annexin B12) and membrane effects are additive. This creates a gradient in water entropy with distance from the surface, with potential consequences for recognition and binding events involving membrane proteins.

The hydration environment of membrane protein annexin B12 in a lipid membrane.

A technique called oriented-sample solid-state NMR can supply information about the water-accessibility of individual residues of membrane proteins in situ, say Gianluigi Veglia and colleagues at the University of Minnesota (A. Dicke et al., JPCB 120, 10959; 2016 – paper here). They’ve used the method to gather this information for the archetypal small transmembrane protein sarcolipin in synthetic bilayers, and find that, as one might expect, there is a relatively smooth gradient of water accessibility with increasing depth within the membrane.

Does the denaturing effect of osmolytes such as urea and guanidinium chloride depend on concentration? Experiments using FRET and SAXS have produced conflicting results, and Robert Best at the NIH and colleagues try to resolve the matter using simulations (W. Zheng et al., JACS 138, 11702; 2016 – paper here). Their test case is the intrinsically disordered protein ACTR, for which they calculate the chain swelling and radius of gyration as a function of denaturant concentration. The protein does indeed swell to a degree proportional to concentration, but the researchers show that nevertheless the structural changes are consistent with SAXS results that appear to show no change in radius of gyration. The denaturant effects operate by a direct mechanism of weak association with the protein.

What is there is a mixture of denaturants such as urea and stabilizing osmolytes such as TMAO? It seems that TMAO can counteract urea’s effects, but how exactly is the hydrophobic interaction affected in that environment? Indrajit Tah and Jagannath Mondal at the Tata Institute in Hyderabad look into that question via simulations of a model hydrophobic polymer and polystyrene (JPCB 120, 10969; 2016 – paper here). In contrast to what is observed with proteins, for the hydrophobic polymer TMAO actually reinforces the destabilizing effect of urea. This seems to be due to the different direct interactions with the polymer chain: for proteins, TMAO is excluded from the surface while urea remains bound, whereas for the hydrophobic polymer both osmolytes may individually bind to the surface. In this latter case, exclusion of TMAO by urea in the mixed solution depletes the opportunities of TMAO to stabilize the collapsed state.

Guangzhao Zhang and colleagues at the South China University of Technology in Guangzhou look at the same denaturant-inhibiting effect of betaine, this time with lysozyme as the model protein (J. Chen et al., JPCB 120, 12327; 2016 – paper here). Using proton NMR, they conclude that in this case betaine interacts directly with urea to form dimers, removing the urea from the protein surface where otherwise it interacts directly to stabilize the denatured state.

Ion-specific “Hofmeister” effects on protein stability and aggregation are still not fully understood at the molecular level. Simon Ebbinghaus at the Ruhr University of Bochum and colleagues seek insights through thermodynamic (DSC) measurements on bovine ribonuclease A (M. Senske et al., PCCP 18, 29698; 2016 – paper here). By measuring ion effects over the whole temperature- and concentration-dependent landscape of protein stability, they find a very complicated picture, due to a complex interplay of contributions. At low concentrations, electrostatic (non-ion-specific) effects dominate, but at higher concentrations there is ion specificity. It’s hard (for me, anyway) to summarize the findings, but I believe it if fair to say that the authors are seeking a unified molecular picture that helps to explain not only ion effects but also those of non-electrolyte cosolutes on protein stability, in terms of a balance between entropic and enthalpic contributions to the excess free energy.

A belated addition from the Bochum group: Yao Xu and Martina Havenith have provided a nice summary of recent work using THz spectroscopy to look at ps-scale collective hydrogen-bond dynamics in the hydration shells of proteins, and the role that they play in molecular-recognition processes (JCP 143, 10.1063/1.4934504; 2015 – paper here).

A further use of THz spectroscopy to investigate hydration is reported by Y Ogawa and colleagues ay Kyoto University (K. Shiraga et al., Appl. Phys. Lett. 106, 253701; 2016 – paper here). Their aim is simply to get some bulk estimate of how much of the water in a cell (they use HeLa cells) has retarded dynamics – a question explored here in the context of the now more or less obsolete notion of “biological water” in cells. Their answer: about a quarter of the total water content has reorientational dynamics slower than the bulk, presumably because of its involvement in biomolecular hydration. This is more than the 10-15% reported previously in prokaryotes and human red blood cells. But of course the absolute numbers must depend on where one places the thresholds in a dynamical continuum.

The voltage-dependent proton transport channel Hv1 is implicated in diseases ranging from cancer to some forms of brain damage. This makes it a potential drug target, and some inhibitors seem to expel bound water from the pore when they bind. Because this water forms intermittent hydrogen-bonded clusters and water wires, it seems likely that there’s a hydration-related entropic contribution to the binding free energy. With this in mind, Mike Klein at Temple University and colleagues have used modeling and simulations to look at water fluctuations in the pore, so as to identify potential binding sites (E. Gianti et al., PNAS 113, E8359 2016 – paper here). Their analysis reveals two such sites: one the binding site known already, another at the outlet of the proton pathway, both of them associated with maximal fluctuation, apparently on the brink of a drying transition, and in locations where replacement by a hydrophobic ligand is optimal. The researchers say that the second, new site should therefore also be considered as a locus of drug design.

The same group has also considered the mechanism of proton transport (S C. van Keulen et al., JPCB 10.1021/acs.jpcb.6b08339; 2016 – paper here). It’s been suggested previously that this occurs via Grotthuss hopping along a water wire. But the results of these quantum/molecular mechanics simulations suggest that instead the proton hops between three acidic residues via mediating water molecules.

How a proton (circled in purple) makes its way down the channel of Hv1: via a series of water-mediated hops between acidic residues.

More proton transport: on the basis of FTIR experiments, Udita Brahmachari and Bridgette Barry at Georgia Tech say that a crucial stage of the oxygen-forming S-cycle of photosynthesis involves the insertion of a proton into a hydrogen-bonded water network (JPCB 120, 11464; 2016 – paper here). Thus bound water here acts as a catalytic proton acceptor and donor.

The catalytic water network in the S3-S0 stage of the photosynthetic cycle.

Jonathan Nickells at Oak Ridge and coworkers have characterized the general hydration environment of green fluorescent protein using neutron scattering spectroscopy to probe the dynamics (S. Perticaroli et al., JACS 10.1021/jacs.6b08845; 2016 – paper here). These dynamics are slowed over just two hydration shells: by a factor of 4-10 in the first shell and 2-5 in the second.

The hydration of GFP.

In comparison, Keiichiro Shiraga and colleagues at Kyoto see dynamical perturbations out to three or four hydration layers (a distance of around 8.5 Å) around albumin, based on THz spectroscopy (K. Shiraga et al., Biophys. J. 111, 2629; 2016 – paper here). They say that the hydrogen-bond network in the hydration layers seems to be less defective than that in the bulk, even though there seems to be greater distortion of the network away from tetrahedral.

Meanwhile, Monique Tourell and Konstantin Momot at the Queensland University of Technology have zeroed in on the single-water-molecule bridges that link parts of the peptide chains in collagen (JPCB 120, 12432; 2016 – paper here). Some experimental studies have apparently implied that the waters are “ice-like” in their dynamics, although I’m not clear quite what this is meant to imply. In any event, these MD simulations suggest otherwise: the waters exhibit strongly anisotropic rotation in which a single molecule might flip back and forth many times while remaining resident at the bridging site for more than 100 picoseconds.

When a particle is solvated at the air-water surface, fluctuations of the interface such as capillary waves may contribute to the solvation free energy – the solute might dampen the fluctuations, for example. Kaustubh Rane and Nico van der Vegt set out to quantify this using Monte Carlo simulations (JPCB 120, 9697; 2016 – paper here). They find that the contribution of fluctuations is not negligible in general, and that the dampening effect is a generic one that doesn’t depend on the chemical nature of solute or solvent. However, the strength of the interactions between ions and water will determine the magnitude of the effect of fluctuations, so that one can expect ion-specific propensities towards proximity to the water surface.

Ariel Fernández, now at the Argentine Mathematics Intitute, has been for some time exploring the role of hydrogen-bond frustration in protein-protein and protein-ligand interactions. The idea here is that such frustration contributes to interfacial tension via its effects on “non-Debye” polarization. Now he has studied how “minimal frustration” might guide these molecular assembly processes and be exploited in drug design, potentially enabling a high degree of binding selectivity (FEBS Lett. 590, 3481; 2016 – paper here).

There’s more on the use of hydration information for drug design from Gerhard Klebe of the University of Marburg and colleagues (S. G. Krimmer et al., J. Med. Chem. 59, 10530; 2016 – paper here). They say that optimizing the water layers covering ligands bound to their target – here hydrophobic inhibitors of thermolysin – can boost the enthalpic contribution to binding free energy. MD simulations enabled the prediction of high binding affinity for a series of ligands, one of which then proved to have 50 times better binding affinity than the known (and patented) parent ligand. This is a really nice piece of work, showing that it’s not just trapped or displaced water molecules that are important in drug design but also the final bound-ligand hydration profile.

The hydration structure of the best drug candidate for binding in the hydrophobic pocket of thermolysin.

Nested fullerenes, or “carbon onions”, cluster in water. Adam Makarucha have used MD simulations to look at the size- and shape-dependence of the effect (A. J. Kakarucha et al., JPCB 120, 11018; 2016 – paper here). As one might expect for a hydrophobic surface, there is layering of water at the surface of these particles, and the disruption of the hydrogen-bond network increases with increasing particle size because of the increased shape anisotropy: the tendency for the larger fullerene shells to become faceted with vertices (where the pentagonal rings sit).

To what extent are the bulk properties of pure water altered by confinement? Debates about confinement-induced changes in “water structure” have sometimes tended to overlook generic effects such as layering of a liquid close to a wall. Roland Netz and colleagues at the Free University of Berlin bring some clarity to the problem by using MS simulations to look at changes in the dielectric properties of water due to confinement-induced correlations in the polarization of neighbouring water molecules (A. Schlaich et al., Phys. Rev. Lett. 117, 048001; 2016 – paper here). They find that these effects – specifically an anticorrelation for neighbouing molecules – result in a significantly decreased dielectric response perpendicular (but not so much parallel) to a pair of walls (here consisting of closely packed decanol monolayers) separated by up to a nanometre or so. This behaviour has obvious consequences for, say, water’s ability to screen electrostatic interactions between closely spaced surfaces (of proteins or lipid membranes, say). [There’s also an APS Physics comment piece on this here]

The existence of a quasiliquid layer on the surface of ice below 273 K is now fairly well attested. Mischa Bonn and Ellen Backus of the MPI for Polymer Research at Mainz and their colleagues have studied this layer using SFG and simulations (M. A. Sánchez et al., PNAS 114, 227; 2016 – paper here). They find evidence of a stepwise transition from a single to a double bilayer of water molecules around 257 K. They say that there is evidence for the single bilayer being quasiliquid all the way down to 235 K.

Friday, October 28, 2016

Are hydrophobic protein surfaces like big or small hydrophobes?

It seems to me that a paper on protein denaturation by Michele Vendruscolo at Cambridge, Stefano Gianni at the University of Rome La Sapienza, and their colleagues will repay careful study (C. Camilloni et al., Sci. Rep. 6, 28285; 2016 – paper here). The researchers use MD simulations to model observed NMR shifts during hot and cold denaturation, and thereby to gain insight into transition-state structures, changes in hydration and thermodynamic parameters. This enables them to characterize in some detail the differences between hot and cold denaturation: the former has more secondary structure, being more influenced by hydrophobic interactions. In effect this points to the existence of two alternative folding mechanisms from denatured states. What’s more, water molecules at the protein surface have the same number of hydrogen bonds on average as those in the bulk. This is what theory predicts for small hydrophobes (<1 nm or so), whereas for larger extended hydrophobic surfaces some hydrogen bonding is thought to be inevitably lost. In other words, it seems that the hydrophobicity of proteins must, on account of their complex surface topography and chemical heterogeneity, be considered to be more akin to that of small rather than large hydrophobes.

A detailed look at how protein surface topography and chemistry affects local water organization is described by Tom Kurtzman of Lehman College and coworkers (K. Haider et al., JPCB 120, 8743; 2016 – paper here). They look at the ligand-binding clefts of six structurally diverse proteins and identify circumstances where the constraints on local water structure compromise the enthalpy. These include deep and narrow cavities and ones with weak water-solute interactions, but also sometimes sites with charged residues in which there can be frustration of adjacent water-water interactions. Clearly this kind of information should be useful for designing ligands that bind competitively to a site, but it remains unclear whether one can yet identify generic design features or whether each case must be considered on its own terms. The broader question is perhaps whether the hydration characteristics must be considered to have been selectively fine-tuned or simply a necessary compromise occasioned by conflicting demands on the binding site. That’s to say, are the hydration environments each individually ‘functional’ or at least partly an epiphenomenon?

One of the clearest cases I can remember seeing for the coupling of hydration structure with protein motions and function is supplied by Tomotaka Oroguchi and Masayoshi Nakasako at Keio University (Sci. Rep. 6, 26302; 2016 – paper here). They have looked at the hexameric multi-domain protein glutamate dehydrogenase (GDH) using MD simulations and AFM. The opening and closing of a hydrophobic pocket HS1 are accompanied by wetting and drying of the pocket, while binding and unbinding of water molecules in a hydrophilic crevice HS2 accompany changes in its length. These two changes in hydration are coupled, creating a kind of hydration-driven mechanism for large-scale conformational change in GDH.

Drying and wetting of the hydrophobic pocket HS1 accompanying the opening and closing of the cleft.

Conformational changes in the GDH protein due to coupled hydration changes at the sites HS1 and HS2.

The coupling of hydration change to large-scale protein dynamics is also the subject of an experimental study by Keisuke Tominaga and colleagues at Kobe University using dielectric spectroscopy at THz frequencies (N. Yamamoto et al., JPCB 120, 4743; 2016 – paper here). They look at lysozyme in the solid state under different hydration conditions, and see two relaxational modes. They attribute the faster of them, with a ~20 ps relaxation time, to coupled water-protein motion: the mode is primarily due to hydration water dynamics, but the hydration water “drags” with it the hydrophilic groups at the protein surface. The slower (~100 ps) mode might be due to motions of the amino-acid side-chains induced by hydration. Looking at the temperature dependence of the spectra, the authors also see a signature of the familiar dynamical transition around 200 K.

The same issue is explored by Dongping Zhong and colleages at Ohio State University using femtosecond spectroscopy of tryptophan relaxation (Y. Qin et al., PNAS 113, 8424; 2016 – paper here). They too see coupling between hydration water and protein side-chain dynamics which slows down the water reorientational relaxation at the interface relative to the bulk. (They study DNA polymerase IV.) Mutational studies and MD simulations imply that causation here goes in the direction of the protein side-chain fluctuations being slaved to the cooperative dynamics of the hydration water.

A closer look at the dynamics of hydration water is provided by Peter Bolhuis of the University of Amsterdam and colleagues, who compare MD simulations with femtosecond IR spectroscopy of the hydration of bovine α-lactalbumin, both in its native and a misfolded state (Z. F. Brotzakis et al., JPCB 120, 4756; 2016 – paper here). The water relaxation times here are typically of the order of tenths of to several picoseconds; ‘slow’ waters have relaxation times > 7 ps, some as much as 20 ps. These waters tend to be located in concavities on the protein surface and make fewer hydrogen bonds with surrounding waters than do molecules in the bulk. Moreover, waters near hydrophobic groups tend to be slower on average than those near hydrophilic groups. But although misfolding exposes more of the hydrophobic surface, it also means that these hydrophobic regions are less concave, and so the water dynamics is somewhat faster on average and there are fewer of the “ultraslow” sites.

Water reorientational decay times seen in simulations of native (left) and misfolded (right) bovine α-lactalbumin.

A somewhat comparable exercise is conducted for B-DNA by James Hynes and Damien Laage at the ENS Paris and colleagues (E. Duboué-Dijon et al., JACS 138, 7610; 2016 – paper here). And there are some commonalities: while the hydration water is generally rather slower to reorient than in the bulk, the waters confined in the narrow minor groove are much more significantly retarded (relaxation times 30-85 ps). Moreover, there is considerable heterogeneity, and some of this comes from coupling of the macromolecular fluctuations with the water dynamics, especially in the minor groove. In other words, there does not seem in this case to be slaving of biomolecular dynamics to those of the solvent, but more or less the reverse.

Water reorientational times on the minor and major grooves of the B-DNA dodecamer (CGCCAATTCGCG)2

Lorna Dougan at Leeds and colleagues have found evidence of a low-density form of water at low temperatures (285-238K), which might be related to the putative phase transition separating low- and high-density liquids in the metastable regime (J. J. Towey et al., JPCB 120, 4439; 2016 – paper here). They keep the water liquid by mixing it with the cryoprotectant glycerol. Neutron scattering and simulation show that at low temperatures the mixture segregates at the nanoscale, and the water nanophase has greater tetrahedral ordering than the bulk.

Predicting protein structure from sequence data often draws on information on homologous structures or fragments from the Protein Data Base. But such homologies cannot always be spotted, or might not be present in the database, or might not be reliable. Peter Wolynes and colleagues at Rice have developed a scheme for predicting structures ab initio, without bioinformatics input, using what they call the atomistic, associative memory, water mediated structure and energy model (AAWSEM) (M. Chen et al., JPCB 120, 8557; 2016 – paper here). This uses coarse-grained simulations at the whole-protein level while drawing on atomistic simulation of fragments – and crucially, incorporates water-mediated interactions in the folding process. It’s a smart approach to the folding problem that draws on the biological reality – the fact that protein folding is funneled to make it evolutionarily robust to small variations in sequence – rather than brute-force number-crunching.

Water mediation is thought to be important too for the aggregation of amyloid fibrils. Samrat Mukhopadhyay and colleagues at the Indian Institute of Science Education and Research in Mohali have used time-resolved fluorescence measurements on the human prion protein (PrP) to investigate how (V. Dalal et al., ChemPhysChem 17, 2804; 2016 – paper here). They find that water hydrating the amyloid-competent oligomers has mobility retarded by three orders of magnitude relative to the bulk, perhaps because of entrapment in the collapsed polypeptide chains. They say that this water might create a hydrogen-bonded network that stabilizes the partly unfolded, molten oligomer conformation and acts as a scaffolding for the assembly of oligomers into fibrils.

Proposed role of ordered water molecules in the misfolding and amyloid formation of PrP – and in protein misfolding diseases more generally.

Antiviral drugs against influenza B could work by blocking the proton-conducting channel BM2, but no such have yet been devised. Mei Hong at MIT and colleagues have used NMR to investigate the mechanism of proton transport in BM2 and the role of hydration, and to elucidate the differences with AM2 from influenza A (J. K. Williams et al., JACS 138, 8143; 2016 – paper here). The His19 residue in BM2 remains unprotonated to lower pH than the corresponding His 37 in AM2, but increasing channel hydration in acidic conditions seems to enhance proton transport to His 19 from water molecules.

Why trehalose acts as a cryoprotectant of protein structure still isn’t fully understood. Jan Swenson and coworkers at Chalmers University of Technology in Göteborg try to develop a comprehensive picture by looking at how trehalose affects the protein glass transition, denaturation temperature, and solution viscosity (C. Olsson et al., JPCB 120, 4723; 2016 – paper here). They study the myoglobin-trehalose-water system using DSC and viscometry. In short, their results seem to exclude the picture in which trehalose displaces water in the solvation shell; on the contrary, they suggest that the protein retains one or two layers of water within a stabilizing water-trehalose matrix. This would be consistent with an apparent lack of coupling between the trehalose-water matrix dynamics and the stability of the native protein.

Schematic of the interactions between water, trehalose and protein.

That picture of a lack of direct interaction between trehalose and proteins – the disaccharide is in fact preferentially excluded from the protein hydration layer – is also the general context for an experimental study by Christina Othon of Wesleyan University in Connecticut and colleagues of trehalose bioprotection (N. Shukla et al., JPCB 120, 9477; 2016 – paper here). Using ultrafast fluorescence spectroscopy for two fluorescent probes, they see a slowdown of water reorganizational dynamics at relatively low trehalose concentrations (0.1-0.25 M, well below the vitrification threshold). At these concentrations, there is around 7 water layers between osmolyte molecules. These results therefore support an indirect mechanism for cryoprotection. Sucrose has much the same effect, but less markedly, the researchers say.

The interaction between two hydrophobic particles in water is generally attractive: this is simply the (water-mediated) hydrophobic effect. But Alenka Luzar and coworkers at Virginia Commonwealth University show that this interaction can become repulsive (B. S. Jabes et al., JPC Lett 7, 3158; 2016 – paper here). Such repulsion has been seen before in simulations of fullerenes and carbon nanotubes in water, and has sometimes been attributed to specific structural changes in the water. But Alenka and her colleagues show that it can be explained purely as a geometric effect of the thermodynamic cost of formation of a liquid-vacuum interface bridging the hydrophobic particles (in these calculations, pure and propyl-terminated graphitic nanoparticles) when drying occurs in the intervening space. This process can be modeled with a straightforward, bulk-like Young-type calculation of the surface free energies.

Nanoconfinement effects on water structure and properties are investigated by Vrushali Hande and Suman Chakrabarty of the National Chemical Laboratory in Pune through simulations of water inside reverse micelles and water-in-oil nanodroplets (Phys. Chem. Chem. Phys. 18, 21767; 2016 – paper here). For the reverse micelles the interface is (negatively) charged, and the deviations from bulk-like behaviour are longer-ranged for orientational order than they are for translational ordering. These effects are far less pronounced for nanodroplets in oil, where the interface is hydrophobic, indicating that electrostatic influences on the hydrogen bonding are more pronounced than spatial confinement per se.

Also on nanoconfinement: quite why water has an enhanced mobility in carbon nanotubes remains a matter of some debate. Using IR spectroscopy, Pascale Roy at the Synchrotron Soleil in Gif-sur-Yvette and colleagues suggest that it may be due to unusually “loose” hydrogen-bond networks among water molecules inside the nanotubes (S. D. Bernadina et al., JACS 138, 10437; 2016 – paper here). They look at nanotubes with diameters of 0.7-2.1 nm, in which the water varies from single-file chains to multilayers, and find a spectroscopic signature of “loosely bonded water” in all cases – in the latter seeming to correspond to waters in the outer layers with dangling OH bonds pointing towards the nanotube walls.

The distance dependence of the hydrophobic force between two hydrophobic walls is investigated in MD simulations by Biman Bagchi and colleagues at the Indian Institute of Science in Bangalore (preprint arxiv.1608.04107). They find a bi-exponential force law, with correlation lengths of 2 nm and 0.5 nm, and a crossover close to 1.5 nm. This behaviour is mimicked by the tetrahedral order parameter, but I’m not entirely clear what the authors’ mechanistic explanation is.

Of course, the issue with many studies of this kind is that your results might only be as good as your model. Angelos Michaelidies and colleagues at UCL offer an overview of the extent to which density-functional theory supplies a good description of water, from small clusters to the bulk (M. J. Gillan et al., JCP 144, 130901; 2016 – paper here). In particular they consider how well different functional forms of exchange-correlation terms perform, and what role many-body terms play. Looks like essential reading for anyone using DFT to model aqueous systems.

Many-body effects are also central to a study by Shelby Straight and Francesco Paesani at UCSD of influences of water’s dipole moment on the hydrogen-bond network of pure water (JPCB 120, 8539; 2016 – paper here). They use simulations to predict the infrared spectra of HOD in H2O, and in particular the shape of the OD stretch. They find that the calculated spectral diffusion of this vibrational frequency depends rather strongly on exactly how one truncates a many-body expansion of the water dipole.

How effectively can hydration be described with a coarse-grained model? Bill Jorgensen and colleagues consider the performance of one attempt to balance accuracy and speed that mixes all-atom and coarse-grained descriptions – the so-called AAX-CGS model, in which all-atom solutes are solvated with coarse-grained water (X. C. Yan et al., JPCB 120, 8102; 2016 – paper here). The approach works well for hydrophobic and halogenated alkane solutes, less so for those that are more polar or engage in hydrogen bonding (amines, alcohols). But the efficiency of the calculations beats that of all-atom simulations by about an order of magnitude or more.

Why are ion hydration free energies asymmetric with respect to ion charge? Rick Remsing and John Weeks investigate that question using an analytical model for calculating hydration free energies that involves gradually “turning on” the ion-solvent Coulomb interaction (JPCB 120, 6238; 2016 – paper here). This enables them to see why the Born solvation model fails to capture the asymmetry: in short, it works well enough for slowly varying Gaussian charge distributions but not for the abrupt, delta-function-like distributions in ion cores. Only in the latter case is the asymmetry in response to ion charge recovered.

Friday, May 27, 2016

Dewetting and its origins

Several thermophilic proteins are known to have internal water-filled cavities, and some are known to denature when these cavities are emptied. Could this internal water be the secret to their thermal stability? Fabio Sterpone in Paris and his colleagues have investigated that question by using MD simulations to calculate the hydration free energy of buried water for several homologous mesophilic and thermophilic proteins (D. Chakraborty et al., JPCB 119, 12760; 2015 – paper here). Their findings support that main contention: the buried water contributes favourably and significantly to the stability of the thermophilic proteins. This could therefore offer a strategy for designing proteins robust against high temperatures.

Protein folding is studied with proton NMR by Francesco Mallamace, Gene Stanley and their collaborators (F. Mallamace et al., PNAS 113, 3159; 2016 – paper here). In particular, they aim to elucidate the role of water in the folding process, taking lysozyme as the paradigmatic example. They examine the evolution of hydrophilic (amide NH) and hydrophobic (methyl and methine CH) groups as folding proceeds, both for a reversible unfolded intermediate state and the irreversible denatured state. Hydrogen bonding between amide groups and internal water seems to play a crucial role: water acts as a kind of “glue” between buried amide and carbonyl groups, while the increased mobility of hydrophobic groups as they form clusters in the folded state compensates for the loss of configurational entropy that this entails.

Studying the binding and unbinding kinetics of cavity-ligand interactions could offer valuable insights into the efficacy of drugs and drug candidates. But it’s tough to do using MD simulations, because the timescales are often too long. Bruce Berne at Columbia and his colleagues have developed a “metadynamics” scheme that allows MD to probe timescales of not just many seconds but an hour or so (P. Tiwary et al., PNAS 112, 12015; 2015 – paper here). They examine a prototypical ligand–cavity system: a fullerene in a simple hydrophobic pocket, with explicit water. They find that binding involves an abrupt dewetting transition when the fullerene is constrained to move only along the central axis of symmetry of the system, but that dewetting is continuous, and binding is 20-fold shorter-lived (around 200s), when this steric constraint is removed.

Hydration of a fullerene in a simple binding cavity.

Abrupt dewetting has been observed between two planar hydrophobic surfaces at small separations, where it is simply capillary evaporation by another name. But Matej Kanduc and Roland Netz at the Free University of Berlin find that it can also occur between a hydrophobic and mildly hydrophilic surface, if they are both polar but electrically neutral (PNAS 112, 12338; 2015 – paper here). This leads to dry adhesion of the dissimilar surfaces. Kanduc and Netz construct the full phase diagram for any pair of surfaces, showing that dry adhesion can happen even for appreciable hydrophilicity of one surface (say, contact angle of 45 degrees) if the other is markedly hydrophobic (contact angle around 120 degrees).

The phase diagram of attractive/dewetting regimes for two planar surface of different contact angle.

Masataka Nagaoka at Nagoya University and colleagues look at another dewetting process, which takes place in the binding pocket of thrombin when it binds a ligand (I. Kurisaki et al., JPCB 119, 15807; 2015 – paper here). The process is quite complicated. These MD simulations indicate that, following the establishment of a hydrogen-bonding interaction between the substrate and an Asp group in the pocket, the water is gradually removed from the pocket, going not into the bulk phase but into a water channel within the protein – which thus turns out to have a functional role.

The water channel of thrombin.

The importance of density fluctuations at the interface in nanoscale (as opposed to atomic-scale) hydrophobic interactions driven by drying transitions has been asserted for some time now by David Chandler and his colleagues. Phillip Geissler and Suriyanarayanan Vaikuntanathan recently developed this idea by showing that the Lum-Chandler-Weeks drying-induced hydrophobic attraction needs to pay close attention to capillary waves (Phys. Rev. Lett. 112, 020603; 2014). Now they and their coworkers have put this theory to the test in a coarse-grained lattice model (S. Vaikuntanathan et al., PNAS 113, E2224; 2016 – paper here). Despite the relative simplicity of the model, it seems to capture very efficiently the nanoscale density fluctuations and their relevance for hydrophobic hydration in the case of a hydrophobic solute particle at the air-water interface and a pair of nanoscale hydrophobic plates. In both cases the “minimalistic” model agrees very well with detailed atomistic simulations, suggesting that it succeeds in modeling the basic physics of these situations. What seems striking here, I think, is how this long-problematic issue of hydrophobic hydration seems to be turning back towards the “old-style” basic physics of wetting and inhomogeneous fluids (compare, for example, R. Evans & M. C. Stewart, J. Phys. Condens. Matt. 27, 194111; 2015; R. Evans & N. B. Wilding, PRL 115, 016103; 2015).

Another coarse-grained description of hydration is offered by Bill Jorgensen at Yale and colleagues (X. C. Yan et al., JPCB jpcb.6b00399; 2016 – paper here). They use techniques I’m frankly not familiar with to couple a coarse-grained approach with all-atom solute models, and find that the resulting multiscale simulations compare acceptably with the all-atom case while achieving a 7-30-fold reduction in computational cost.

And on the same track, Kenichiro Koga at Okayama and colleagues report a mean-field approximation for inhomogeneous liquids to study hydrophobic hydration, specifically the solvation of methane at the air-water interface (K. Abe et al., JPCB 120, 2012; 2016 – paper here). The approach here is to consider two steps in the solvation process: creation of the cavity for a hydrophobic solute, and insertion of the solute itself.

Ions modify hydrophobic interactions in an ion-specific manner, familiar as Hofmeister effects. But to what extent do these influences depend on the arrangements and mobility of the ions? Izabela Szlufarska at the University of Wisconsin and colleagues examine that question using MD simulations of hydrophobic interactions between a nonpolar surface and nonpolar or amphiphilic nanorods mimicking β-peptides (K. Huang et al., JPCB 119, 13152; 2015 – paper here). They compare the cases of free ions in solution, and ionic groups tethered to the rods both randomly and in organized spatial arrangements (such as a row down one side). Free ions generally strengthen hydrophobic interactions according to the conventional ion-specific series, in a manner that is correlated with (and assumed to be due to) fluctuations in water density close to the surface. But these interactions can be switched on or off by different arrangements of the immobilized ions, and the ion-specific effects now don’t follow the usual Hofmeister ranking. The authors analyse the findings in a dynamical picture, concluding that “Our analysis of the structure and dynamics of water near the hydrophobic nanorod shows that dynamics is a better indicator of the specific ion effect than the static water structure.” Is this perhaps an emerging consensus for such situations?

The nanorods studied by Huang et al.: cyan groups are nonpolar, yellow are immobilized ions.

Hydration water around intrinsically disordered proteins is more mobile than that around globular proteins, according to simulations by Pooja Rani and Parbati Biswas at the University of Delhi (JPCB 119, 13262; 2015 – paper here). They find that both the translational and rotational diffusion are relatively greater for IDPs. In an earlier study, they found that the residence times of hydration waters for IDPs are relatively long (JPCB 119, 10858; 2015) – but that work doesn’t contradict this, they argue, because the enhanced mobility doesn’t actually help the water molecules leave the hydration layer.

Water wires are essential for transporting protons through the pores of the chloride/proton exchanger ClC-ec1, a member of the CLC superfamily of proteins that exchange protons for halides and other anions across cell membranes. That process is elucidated in detail in simulations by Emad Tajkhorshid at UIUC and colleagues (T. Jiang et al., JACS 138, 3066; 2016 – paper here). They examine how the water wires formed in the presence of chloride bound in the central anion binding site differ from those in the presence of fluoride, nitrate and thiocyanate, giving the channel its anion specificity. For fluoride and nitrate there are only “pseudo water wires” separated by the intervening anion, which can’t sustain proton transport; for thiocyanate there are none at all.

Water wires and their disruption/absence for various anions in the channel of the chloride/proton exchanger ClC-ec1.

Is the air-water interface acidic or basic? In other words, do hydrated excess protons or hydroxide ions have an affinity for the water surface? Simulations and experiments have sometimes seemed to give conflicting results, making this a point of controversy. Greg Voth, now at the University of Chicago, and his coworkers reported several years ago that simulations showed protons having an affinity for the interface. They now confirm this finding using state-of-the-art reactive molecular dynamics (Y.-L. S. Tse et al., JACS 137, 12610; 2015 – paper here). The (weak) preference of the hydrated proton of the water surface, they say, is enthalpically favoured and reduces the loss of hydrogen-bonding there. The hydroxide ion, meanwhile, is repelled from the interface, also for enthalpic reasons.

Small peptides don’t sample all of the conformational space theoretically available to them. Brigita Urbanc and colleagues at Drexel University in Philadelphia wonder if hydration structure has something to do with this (D. Meral et al., JPCB 119, 13237; 2015 – paper here). Using MD simulations, they show that the so-called polyproline II-like (pPII) and β-strand conformations that are prominent for GXG peptides (with X a “guest” residue) have different hydration structures, the former being enthalpically stabilized and the latter entropically. In general the nature of backbone hydration in the pPII conformations is clathrate-like, and differs for different X depending on the residue’s ability to template this structure.

It’s looking ever more as though water has an important, even pivotal role in the assembly of the β-amyloid aggregates implicated in neurodegenerative diseases. Nadine Schwierz at UC Berkeley and coworkers report that it drives the growth of these fibrils (N. Schwierz et al., JACS 138, 527; 2016 – paper here). Their simulations suggest that solvent entropy is the main driving force, and that assembly involves collective water motions. As two β strands lock together, motion of the intervening water is significantly retarded, and there is an entropic gain when this water is removed to create a dry binding interface.

The hydrophobic interaction is still challenging to understand intuitively. That’s emphasized by a report by Lawrence Pratt at Tulane University and colleagues showing that, for argon-argon interactions in water, the “hydrophobic bond” is actually weakened by including solute attractive interactions (M. I. Chaudhari et al., JPCB 120, 1864; 2016 – paper here). They treat the situation using local mean field theory, and I think (but am not sure) that ultimately the reason for this result is that in effect the solute attractive interactions benefit solvation more than they do aggregation.

Might water networks in a protein binding site be amenable to a thermodynamic treatment that considers them a kind of loose, composite ligand? That seems to be in essence what Gregory Ross at the University of Southampton and colleagues are attempting to enable (G. A. Ross et al., JACS 137, 14930; 2015 – paper here). They have modified the grand canonical MC simulation technique to allow efficient calculation of the binding energy of an entire hydrogen-bonded water network, as well as to estimate the individual water-molecule affinities and the degree of cooperativity between them. They propose that the method might allow the incorporation of water networks into rational drug design.

How to handle long-ranged forces has, as John Weeks at Maryland and colleagues put it in a paper in PNAS (R. C. Remsing et al., PNAS 113, 2819; 2016 – paper here), “plagued theory and simulation alike”. They offer a solution: a way to incorporate long-ranged interactions that uses only short-ranged potentials. They demonstrate it for the case of hydration of ionic and hydrophobic species. It relies on local molecular field theory, in which the interactions are accommodated by finding a simpler, single-particle “mimic system” that can furnish an effective field that takes care of averaged “far-field” interactions in the full system under study.

Different types of antifreeze proteins have different mechanisms of action, according to Ilja Voets at the Eindhoven University of Technology and colleagues (L. L. C. Olijve et al., PNAS 113, 3740; 2016 – paper here). They have measured both the thermal hysteresis and the ice recrystallization inhibition activity for a range of AFPs, and find no correlation between the two. Both of these properties have been considered to be features of antifreeze activity, which the researchers attribute to binding to different ice planes. They conclude that antifreeze activity is many-factored, and that applications and rational design of AFPs “requires a tailored optimization to the specific purpose.”

More debate on the putative liquid-liquid transition in metastable water: Gyan Johari and José Teixeira argue that the thermodynamic arguments linking this transition to that between low- and high-density amorphous ice don’t stand up (JPCB 119, 14210; 2015 – paper here). They conclude that “The available data show that HDA is not a glass, and the presumed HDL and LDL are not normal liquids. If that is accepted, the HDL−LDL fluctuations view, the two-liquid model, and the virtual liquid−liquid phase transition would all be consequences of a false premise.” I look forward to the next round…

Two quite different forms of metastable (quasi-)liquid water are reported by Gen Sazaki and colleagues at Hokkaido University. They have previously reported such phases on the surface of ice (Sazaki et al., PNAS 109, 1052; 2012); one of the liquid phases wets the ice, the other doesn’t. Using advanced optical microscopy, they now show that these surface phases are kinetically, not thermodynamically, stable, and that they form not by the surface melting of ice but by the deposition of supersaturated water vapour on the ice surface (H. Asakawa et al., PNAS pnas.1521607113; 2016 – paper here).

Julio Fernández continues his studies of “frustrated” water at the protein interface (dehydrons, which are topologically deprived of hydrogen-bonding opportunities) with a study of the acid-base chemistry of such water, using quantum-chemical calculations (FEBS Lett. 590, 215; 2016 – paper here). He reports that water surrounding deydrons is proton-accepting, and enables a mechanism for directed Grotthuss transport of protons

Tryptophan fluorescence has been widely used to study protein dynamics and hydration. Feng Gai and colleagues at the University of Pennsylvania report that a tryptophan analogue, 5-cyanotryptophan can be used in the same way but with greater sensitivity (B. N. Markiewicz et al., JPCB 120, 936; 2016 – paper here). They show that, among other things, the new probe molecule allows them to distinguish two differently hydrated environments in a folded protein (Trp-cage).

Whether or not the result reported by Ruth Livingstone and colleagues at the MPI Mainz is relevant to water in biology isn’t clear, but it’s an interesting finding all the same. They use vibrational spectroscopy to look at water interacting with a monolayer of the detergent sodium dodecyl sulfate at the water surface, and see two distinct types of water present (R. Livingstone et al., JACS 137, 14912; 2015 – paper here). Close to the anionic head groups the waters have localized O-H stretches, but further below the monolayer there is a delocalized O-H mode. These two modes are coupled and can exchange energy. The question is obviously whether the same applies for lipid assemblies in vivo.

Tuesday, March 1, 2016

Why DNA hydration is different from that of proteins

Well, it seems that post-Christmas catching up now takes until March… Hopefully normal service hereafter.

Why trehalose confers protection against urea-induced denaturation of proteins is investigated by Subrata Paul and Sandip Paul of the Indian Institute of Technology in Guwahati using MD simulations for a protein analogue (N-methyl acetamide, NMA) (JPCB 119, 9820; 2015 – paper here). They find that direct interactions are the key. NMA hydration water is displaced by hydrogen-bonded urea, but the addition of trehalose only modestly decreases the urea density close to the amide. Rather, trehalose molecules largely replace water molecules in the hydration shell (so that trehalose and urea bind to NMA simultaneously). For proteins this would reduce the ability of water to solvate exposed backbone in the denatured state.

Another structure-stabilizing osmolyte, trimethylamine N-oxide (TMAO), is studied by Yuki Nagata of the MPI for Polymer Research in Mainz and colleagues (JPCB 119, 10597; 2015 – paper here). Their ab initio simulations look at the effect of TMAO on water reorientational dynamics, and show that these are retarded significantly close to the TMAO’s oxygen atom due to its hydrogen bonding with water – a result not seen using a simpler force-field model that represents the oxygen as a single point charge. Since TMAO is generally excluded from protein surfaces, its stabilizing influence seems in this case to be an indirect effect, for which the authors say this interaction with water seems likely to be important. These conclusions are supported by the work of Gerhard Schwaab and colleagues at Bochum, who have used THz/FIR and Raman spectroscopy to look at the solvation dynamics of TMAO (L. Knake et al., JPCB 119, 13842; 2015 – paper here). They too find strong hydrogen bonding between TMAO and water, which supports an indirect mechanism for its biological effects.

How proteins denature in pure water as a result of low temperature or high/low pressure is still not entirely clear – in particular, are the mechanisms of these two processes essentially the same or distinct? That questions is addressed by Valentino Bianco and Giancarlo Franzese at the University of Barcelona using MC simulations of a coarse-grained protein model (represented as a self-avoiding hydrophobic or mixed hydrophobic/hydrophilic chain) with explicit water solvent (Phys. Rev. Lett. 115, 108101; 2015 – paper here). They argue that both denaturation processes involve a balance of free energies between hydration and bulk water. For cold denaturation, this balance favours the unfolded protein conformation for energetic reasons relating to the increasing stabilizing effect of water-water hydrogen bonds in the hydration shell. High-pressure denaturation, meanwhile, is driven by changes in local water density near the unfolded protein, giving a dominant PV contribution to the free-energy change of denaturation. And low-pressure denaturation is enthalpically driven, again by changes in the number of water-water hydrogen bonds in the hydration shell. In sum, the authors say, “For [all] these mechanisms is essential to take into account how the protein-water interactions affect the stability of the water-water HB and the water density in the hydration shell.”

A strategy for attacking antibiotic resistant bacterial pathogens that has been afforded increasing attention recently involves disabling not the mechanisms by which they synthesize key cellular components but those that contribute to virulence. One such target is a dehydratase enzyme DHQ1, present in several typical “superbugs”. Concepción González-Bello at the University of Santiago de Compostella and coworkers describe an inhibitor containing an ammonium group that binds to DHQ1 without relying on the reactive epoxide functionality of previous such drug candidates (C. González-Bello et al., JACS 137, 9333; 2015 – paper here). It binds to and covalently modifies the enzyme’s active site thanks to hydrogen-bonding to a single water molecule, while a second conserved water molecule participates in the reaction that leads to covalent modification.

Another water-assisted enzymatic mechanism is described by Ana-Nicoleta Bondar of the Free University of Berlin and colleagues. Using MD simulations, they look at the proton-transfer protein PsbO, a subunit of photosystem II, and find low-mobility water molecules close to its surface that form part of a water-carboxylate network that could facilitate proton transport (S. Lorch et al., JPCB 119, 12172; 2015 – paper here). Some of these waters might, they say, also assist the docking of PsbO into the PSII complex.

The water-carboxylate network on the surface of the proton-transfer protein PsbO.

In contrast to the often slow dynamics of “functional water” at protein surfaces, Songi Han at UCSB and colleagues report anomalously fast water diffusion near DNA surfaces (J. M. Franck et al., JACS 137, 12013; 2015 – paper here). They use Oberhauser effect dynamic nuclear polarization (ODNP) to look at water translation dynamics along the backbone, which entails adding spin centres along a DNA backbone to act as “reporters”. Anomalously fast here means bulk-like, in contrast to the retarded diffusion of most of the hydration water, suggesting that the DNA-water interactions are only weak for a significant proportion of the hydration shell. Such water is relatively easily displaced when proteins bind to the DNA. Thus, in comparison to proteins, DNA would be capable of storing less free energy that can be released entropically in binding interactions – and, the authors argue, makes DNA duplexes less specific in their interactions (outside, of course, of the specificity supplied by the sequence itself). So “the high mobility of the solvation water around DNA may have been tailored to its role in biological function.”

I had the pleasure of meeting Xiao Cheng Zeng of the University of Nebraska in Shanghai in November, and hearing about his interesting work on monolayer and bilayer ices. He and coworkers have recently explored the mechanism by which a preference for potassium over sodium ion transport arises in synthetic hydrophobic organic nanopores, namely the stacked macrocycles reported by Zhou et al. in Nat. Commun. 3, 1-8 (2012), and compare them with carbon nanotubes (H. Li et al., PNAS 112, 108512; 2015 – paper here). At face value the preferential K+ transport is surprising, given that K+ has the larger hydration sphere. But the researchers explain this selectivity (which is also seen in some biological ion channels) in terms of the greater robustness and structure of the Na+ hydration shell. Moreover, the roughness of the interior surface of the organic nanopores lowers the diffusivity of both ions – but more so sodium – relative to smooth-sided carbon nanotubes, with the result that the CNTs offer the highest rate of selective K+ transportation.

Jianzhong Wu at the University of California at Riverside use a combination of thermodynamic measurements (Henry’s constants) and MD simulations to examine how the degree of hydrogen bonding among water molecules changes in hydrophobic hydration shells (J. Kim et al., JPCB 119, 12108; 2015 – paper here). They find that the extent of H-bonding is slightly reduced, and that this is a significant factor in the positive hydrophobic hydration heat capacity. Certainly nothing ice- or clathrate-like here.

How do we assess the hydrophobicity of heterogeneous surfaces, composed of a mixture of hydrophobic and hydrophilic patches? Well, it’s complicated. Damián Scherlis at the University of Buenos Aires and colleagues find in simulations with coarse-grained mW water that there is a rather complex variation of water-droplet contact angle on such surfaces with the amount, distribution and arrangement of hydrophilic (hydrogen-bonding) domains on a hydrophobic surface (M. H. Factorovich et al., JACS 137, 10618; 2015 – paper here). In this case, there may be a nonlinear variation of contact angle with composition, the angle being largest for a particular fraction of hydrophobic species (about 0.6 when randomly mixed). In other words, this mixture can have a larger contact angle than the purely hydrophobic surface. What’s more, this variation depends on the size of the domains: the nonlinearity tends to vanish for hydrophilic domains bigger than about 2 nm, when the behaviour returns to being reasonably well described by the Cassie model. The same conclusions are supported by looking at the desorption pressure of water in nanopores of these compositions. This appears to offer considerable scope for tuning the wettability of surfaces.

And I have just discovered that a paper to which I contributed in a very small way, on the notion of chaotropicity as a way of understanding the limiting tolerances of biofuel-producing microorganisms, was published at the end of last year (J. A. Cray et al., Curr. Opin. Biotechnol. 33, 228; 2015 – paper here). My coauthors are to be credited with everything of real substance in this interesting and potentially useful article.

Wednesday, October 7, 2015

What do you mean, water structure?

Ah, water structure. What do we mean by it? How do we measure it? Elise Duboué-Dijon and Damien Laage revisit this old question with a close look at how various popular order parameters fare in describing the hydration shell of a hydrophobic solute in MD simulations (JPCB 119, 8406; 2015 – paper here). The tetrahedrality, local density, Voronoi cell shape and others are considered, and the correlations between them are in general not terribly strong: they are each tending to measure different things. But in any event, the perturbations around the small hydrophobic solute are rather small relative to the bulk: there is nothing iceberg-like here, nor is there any sign of significant heterogeneity. I think it would be fair to say that, rather than implying that water structure is best defined as “X”, we should conclude that “water structure” is an ill-defined concept. The authors also conclude that angular distortions offer the best measure of fluctuations in water reorientation dynamics.

I sense a meaty story in this one. Nascent membrane proteins emerging from the ribosome are assembled and integrated into the cell membrane with the aid of the translocon, a channel-like complex of proteins within the membrane. This complex has an hourglass shape and is filled with water, and the insertion of membrane proteins here has been considered as a simple process of hydrophobic partitioning. But it’s not so simple, according to Stephen White at the University of California at Irvine and colleagues (S. Capponi et al., PNAS 112, 9016; 2015 – paper here). They have performed MD simulations of the bacterial SecY translocon complex, and find that the water inside is very different from the bulk phase, having retarded rotational dynamics and aligned dipoles: in other words, it is decidedly “anomalous water”, suggesting that the translocon can’t simply be regarded as a protein-conducting pore. So any hydrophobic partitioning is likely to be more subtle than has been supposed, and we need to consider some degree of functional modification of the water properties: as the authors put it, “what is the partitioning free energy of solutes between water in bulk and water in restraining confined spaces?”

“If life can be considered as a massive self-assembly process, water seems to be a major driving force behind it.” There’s a nice way to begin a paper, and it’s how Vrushali Hande and Suman Chakrabarty of the CSIR National Chemical Laboratory in Pune start their simulation study of water ordering around hydrophobic polymers (JPCB 119, 11346; 2015 – paper here). They investigate specifically the notion introduced by Chandler and coworkers of a qualitative change in hydration at a length scale of around 1 nm. This depends, the authors say, on the conformation of the polymer. When it is extended, the tetrahedral ordering of the hydration shell is more or less insensitive to polymer chain length, because of the sub-nanometre scale of hydrogen bonding around the polymer chain. But in a collapsed conformation it’s a different story, with the hydration waters then dynamically coupled to fluctuations of the polymer. All the same, tetrahedral ordering doesn’t provide a strong signature of any order-disorder transition in the hydration layer, at least until chain lengths of around C40. But the authors say that this collapse itself is linked to fluctuations in the solvent in the manner discussed by Chandler et al., which can induce local dewetting.

The open and collapsed states of hydrophobic polymers in water, studied by Hande and Chakrabarty.

What is the state of water close to hydrophilic surfaces? There have been several experimental suggestions that this “interfacial water” has, over a nanoscale thickness, a viscosity several orders of magnitude greater than the bulk (e.g. Jinesh et al., Phys. Rev. Lett. 96, 166103; 2006). Andrei Sommer at Ulm and colleagues have recently argued that this interfacial water can be modified by irradiation with near-IR laser light (A. P. Sommer et al., Sci Rep. 5, 12029; 2015 – paper here). They now suggest that the gradient in viscosity that this would imply might explain why and how the rate of ATP synthesis changes in response to both reactive oxygen species and such irradiation. If ROS increase the hydrophilicity of the membrane in which the ATP synthase is embedded, they say, then this will increase the viscosity further and degrade the efficiency of this rotary device. By the same token, IR light decreases the viscosity and has a contrary effect on ATP synthesis. Note that the argument is only indirectly supported by the experiments described here, which are concerned only with measuring changes in the nanoindentation force for a diamond tip penetrating a water-coated hydrophilic metal surface due to laser irradiation, and interpreting them in terms of viscosity changes in the water film.

The controversy around water’s putative liquid-liquid phase continues. There have already been responses to David Limmer and David Chandler’s suggestion that the metastable LL transition reported in previous theoretical work is just an unequilibrated state that would eventually convert to ice (JCP 135, 134503; 2011 and 138, 214504; 2013). But now in a preprint, Frank Smallenburg and Francesco Sciortino say that, by modifying the bond flexibility of ST2 water, they can continuously tune the LL critical point until it moves into a regime where the liquid is more stable than ice – thereby, they say, negating any kinetic arguments for why this critical point is a phantom of the simulation technique

The degree of covalency of the hydrogen bond in water has been much debated. Thomas Kuhne at Paderborn and colleagues propose that this can be quantified by measuring components of the magnetic shielding tensor of the water hydrogens in NMR (H. Elgabarty et al., Nat. Commun. 6, 8318; 2015 – paper here). They define covalency as the amount of electron density transferred between hydrogen-bonded molecules and the associated stabilization energy, which they calculate in ab initio simulations to be, respectively, around 10 milli-electrons and 15 kJ/mol. They describe a calibration of the relationship between these quantities and the hydrogen magnetic shielding tensor that would enable their experimental determination.

The exchange of amide hydrogens in proteins with water can be used as a measure of protein structuring, flexibility, dynamics, and solvent exposure. But the mechanism by which it happens hasn’t been clear. Filip Persson and Bertil Halle show how even deeply buried parts of the polypeptide chain may become briefly exposed to water by conformational fluctuations (PNAS 112, 10383; 2015 – paper here). Their simulations of the bovine pancreatic trypsin inhibitor are long enough to identify the elusive “open” state by which proton exchange happens: a state, they propose, that requires the N-H hydrogen to be within 2.6 Å of at least two water molecules, and not involved in any intramolecular hydrogen-bonding. As well as the donor water molecule, the second water molecule is needed to solvate and stabilize the transient imidate ion formed after proton extraction from N-H, before it acquires a replacement proton from this second molecule. This “open” state exists for around 100 ps on average in all the amide groups studied here.

Bertil continues to probe conformational dynamics in an NMR study with Shuji Kaieda of water displacement within the cavity of a lipid binding protein (JPCB 119, 7957; 2015 – paper here). Conformational changes act to gate this water release, with fluctuations in a critical part of the protein determining the rate of passage of some highly ordered internal waters. The latter fall into three dynamical classes, with distinct survival times of the order of 1 ns (most of the waters are of this type), 100 ns and 6 μs.

Functionally relevant conformational fluctuations are also studied in a preprint sent to me by Tomotaka Oroguchi and Masayoshi Nakasako of Keio University. Their MD simulations suggest that the functional motions of an enzyme (glutamate dehydrogenase) are dominated by nanoscale wetting/drying transitions of a small number of hydration water molecules in a hydrophobic pocket (HS1) of the active site, along with stepwise association and dissociation of water clusters in a cylindrical hydrophilic crevice (HS2). The interpretation of behaviour at the hydrophobic site is supported by measurements of the catalytic rate of a mutant in which this hydrophobicity is lower. The combination of changing hydration states at the two sites makes the enzyme act as something of a hydraulic machine. This offers a nice illustration of how the vague idea of water-lubricated conformational flexibility in proteins can be united with more precise notions of nanoscale wetting and dehydration transitions.

Snapshots of different wetting states for the hydrpphobic pocket HS1 of glutamate dehydrogenase, along with a heat map relating solvent occupancy of this cleft (Q) to the separation of the “jaws” (d).

The “GDH machine”, driven by changes in hydration states in the hydrophobic (HS1) and hydrophilic (HS2) sites.

Ion channel selectivity is largely determined by electrostatic interactions with charged residues in the channel. But Vicente Aguilella and colleagues at the Universitat Jaume I in Castellón present calculations and simulations which challenge the idea that only solvent-accessible residues near the ion permeation pathway matter (E. García-Giménez et al. JPCB 119, 8475; 2015 – paper here). Looking in particular at bacterial porin OmpF, they say that many other charged residues, including buried ones, may affect the pore selectivity and that the dielectric properties of the protein therefore matter.

How does trehalose protect proteins from urea-induced denaturation? Subrata Paul and Sandip Paul at the Indian Institute of Technology in Assam explore that question via MD simulations of the hydration of the simple protein model N-methylacetamide (JPCB 119, 9820; 2015 – paper here). They find that trehalose displaces urea from the vicinity of the amide, and that amide-water hydrogen bonds are replaced by amide-trehalose H-bonds; thus trehalose will reduce the propensity of water to H-bond with a protein backbone, which would otherwise stabilize the denatured state. The results also largely support the notion that urea denaturation occurs via direct interactions rather than indirect effects on “water structure”.

Here is another introductory remark that encapsulates an issue rather splendidly: “If proteins had evolved to fold in a vacuum, thermodynamic experiments in the laboratory could have been straightforwardly interpreted by statistical energy landscape theory, just as model computer simulations with implicit solvent have been. Instead, the intimate involvement of the aqueous environment in the folding process made the uncovering of the principles of the energy landscape theory of protein folding a convoluted process.” This comes from a paper by Peyer Wolynes and colleagues (B. J. Sirovetz et al., JPCB 119, 11416; 2015 – paper here) in which a new model of protein folding (the associative memory, water mediated, structure and energy model, AWSEM) is used to map out the folding diagram for two proteins and explore hot, cold and pressure-induced denaturation. This model uses a coarse-grained force field that, among other things, captures water-mediated interactions. Using ubiquitin and λ-repressor as the test cases, the work shows that the model can supply a unified description of all of these cases that captures the key features of experimental measurements.

Representative structures of uniquitin in the native and denatured states in the AWSEM.

An interesting model system for studying water wires is described by Mihail Barboiu of the European Institute of Membranes in Montpellier (M. Barboiu et al., JPCB 119, 8707; 2015 – paper here). They look at self-assembled structures of a synthetic bola-amphiphile, which contain transverse pores that are hydrophilic, chiral and can contain helical water wires in which the waters are strongly orientationally ordered. Cations can permeate along these channels, offering a simple analogue of ion conduction through biomolecular water channels (for example, in gramicidin A). The authors say that selectivity of ion transport here is dominated by a subtle balance between the hydration and complexation energies of the ions.

Looking down the chiral water channels in crystals of a bola-amphiphile.

In somewhat related territory, Manish Kumar at Penn State University and colleagues describe a new class of artificial water channels that self-assemble into membrane-like structures (Y.-X. Shen et al., PNAS 112, 9810; 2015 – paper here). They call them peptide-appended pillar[5]arenes, which have a linked arene belt in the middle and short peptides extending above and below it, making a tubular structure. These molecules have been investigated before (summarized in Cragg & Sharma, Chem. Soc. Rev. 41, 597; 2012), but those described here are more hydrophobic and will insert at rather high concentration into lipid membranes, making the membrane water-permeable (3.5x10^8 water molecules per s, comparable to aquaporins). Simulations suggest that the channels seem to fluctuate between filled and empty states of water in a wetting/dewetting transition.

Pillar[5]arenes (A and B), and their insertion into a lipid membrane (C). D shows the water permeability.

Something quite different from my colleague John Hallsworth at Queen’s in Belfast and his coworkers, who ask “Is there a common water-activity limit for the three domains of life?” (A. Stevenson et al., Int. Soc. Microbial Ecol. J. 9, 1333; 2015 – paper here). They report that halophilic Archaea and Bacteria, and some xerophilic fungi, can all sustain viability at water activities as low as about 0.61.Could this point to a common physicochemical/thermodynamic origin for such a limiting value? If so, could there be astrobiological implications? (A good time to be thinking about that!)

Another unusual kind of contribution with prebiotic resonances comes from Atul Parikh of Nanyang Technological University in Singapore and coworkers, who report that giant vesicles filled with sugar solution and subjected to osmotic stress in a bath of lower sugar concentration may undergo damped cycles of expansion and contraction, accompanied by temporary rupture of the vesicle walls (K. Oglecka et al., eLife 3, e03695; 2014 – paper here). In the expanded phase, the vesicles are patchy, due to phase separation of cholesterol and phospholipids in the walls; in the contracted phase they are uniform. The researchers say that this might have offered a useful, even adaptive, response to the microenvironment for early protocells. There’s a nice phys.org story on the work here.

Friday, July 24, 2015

Goodbye to "biological water" (hello water in biology)

Surely an essential read for any reader of this blog is a commentary by Pavel Jungwirth in JPC Lett. (6, 2449; 2015 – paper here) called “Biological water or rather water in biology?” (which I only just realize now I can decide to interpret as an homage to this site – you can see which of these alternatives I prefer!). Pavel expresses the issue perfectly, saying that his piece has two main messages:
“The first one, addressed to biologists and biochemists, who tend to focus their attention primarily to the biomolecules, is that water does matter.”
“The second and arguably more important message is addressed to our community of physical chemists:… Although water… plays a key role in establishing the homeostasis, it is primarily the biomolecule itself which carries the biological function… As physical chemists who naturally tend to understand water better than biomolecules, we may sometimes have a tendency to overemphasize the role of the former at the expense of the latter.”

In particular, Pavel suggests that the term “biological water” be dropped. He is quite right that this kind of terminology risks becoming a deus ex machina, if not indeed a kind of “vital force”, and I’d be happy never to see it again. This also gives me an opportunity to say explicitly that, while this blog aims to focus on all the important, often under-valued and occasionally amazing things that water does in the cell, there should be no doubt that proteins, DNA, lipids and carbohydrates are still the main players.

I mentioned in an earlier post a study by Bob Evans at Bristol a study suggesting an explanation for the enhanced density fluctuations in water near a hydrophobic surface that David Chandler, Shekhar Garde and others have advanced as the driving force behind dewetting transitions. Bob and his coauthor Nigel Wilding at Bath have now published this paper (Phys. Rev. Lett. 115, 016103; 2015 – paper here). They argue that the fluctuations can be regarded as a divergence in the local compressibility associated with the approach to a critical (continuous) drying transition. Frankly, it seems rather splendid to have this phenomenon rooted in a general and well understood physical effect – and moreover one that is not at all specific to water or hydrogen-bonding networks.

This seems to bear directly on what Rick Remsing, Amish Patel, Shekhar and others say in their latest paper on “pathways to dewetting” (R. C. Remsing et al., PNAS 112, 8181; 2015 – paper here). Their MD simulations of water confined in the nanospace between two square hydrophobic plates confirm that it undergoes enhanced density fluctuations that can nucleate a vapour tube connecting the plates of a radius greater than the critical radius needed for spontaneous growth to a dry state, according to standard nucleation theory. This means that the free-energy barrier to dewetting is lower than standard macroscopic theory would predict. That’s very striking and illuminating, but still doesn’t obviously say in itself where those enhanced fluctuations come from – which is what Bob’s paper seems to address. You should talk to each other, chaps! – it seems as though there could even be the prospect of tying this story together once and for all.

Suzanne Zoë Fisher at Los Alamos National Laboratory and colleagues have used neutron scattering and NMR to characterize the details of the proton transfer system in human carbonic anhydrase, in which a water network linked to hydrophilic residues plays a key role (R. Michalczyk et al., PNAS 112, 5673; 2015 – paper here). It’s a nice, thorough study which shows how the environment lowers the pKa of the Tyr7 residue bound to the water molecules.

More water wires in a first-principles simulation study by Daniel Sebastiani at the University of Halle-Wittenberg and colleagues – but this time looking at their transient formation in pure water itself (G. Bekçioglu et al., JPCB 119, 4053; 2015 – paper here). In their calculations they use hydroquinoline as a fluorescent probe to study proton transfer along the wires, and find that wires of up to six or seven water molecules, reaching 1.5 nm or so, may persist for up to a few picoseconds. These might facilitate proton transfer (here between donor and acceptor sites on the probe molecule) by a stepwise mechanism.

Samir Kumar Pal at the Bose National Centre for Basic Sciences in Kolkata and colleagues report a nice model system for studying the dynamical coupling between a macromolecule and its hydration sphere (S. Choudhury et al., JPCB 10.1021/jp511899q – paper here). They have used micelles with different degrees of packing rigidity as model macromolecules, and use FRET, polarization-gated fluorescence anisotropy and quasielastic neutron scattering to look at the dynamics of the micelles and their hydration shells. There is slower water motion around the less flexible micelles, consistent with the standard “slaving” picture of dynamical coupling.

Some water of course may penetrate into amphiphile assemblies of this sort. It’s known that cholesterol reduces the permeability of lipid membranes to water, but it’s not clear why. Bilkiss Issack and Gilles Peslherbe at Concordia University in Montreal have studied the question with MD simulations (JPCB 119, 9391; 2015 – paper here). The results imply that this is a thermodynamic and not a kinetic effect – water diffusion doesn’t vary much with cholesterol concentration, but the free-energy barrier to water penetration through a bilayer does increase with concentration, probably because cholesterol increases the hydrophobicity of the core region.

Pooja Rani and Parbati Biswas at the University of Delhi say that intrinsically disordered proteins have a larger binding capacity for water than do globular proteins (JPCB 10.1021/jp511961c – paper here). What’s more, their MD simulations show more tetrahedral ordering, and slower dynamics, of water around disordered protein segments. In a loose sense this seems consistent with the different water dynamics around IDPs observed by Martin Weik and colleagues using neutron scattering – which they see as a difference in degree rather than in kind.

But modelling IDPs accurately using MD requires better water models, according to Stefano Piana of D. E. Shaw Research in New York and colleagues (JPCB 119, 5113; 2015 – paper here). They say that most simulations produce IDP conformations that are too compact, but that they can do better using a new water potential called TIP4P-D, which includes a better representation of the dispersion forces between water molecules. There’s more optimization still to be done, but it’s presumably possible that such improvements aren’t unique to IDPs, even if they are particularly sensitive to them.

Not unrelated is a study by Yuichi Ogawa and colleagues at Kyoto University of the coil-to-globule transition of a “model peptide”, poly(N-isopropylacrylamide) (K. Shiraga et al., JPCB 119, 5576; 2015 – paper here). They follow changes in the hydration state of the polymer during this conformational switch using attenuated total reflection spectroscopy, which is a new one on me but apparently probes changes in the dielectric response in the terahertz region, providing information about the hydrogen-bond network. The transition to globule form corresponds with a reduction in the average hydration number of each monomer from around 10 to about 6.5, and it seems that these changes happen mostly in hydrophobic regions of the polymer. The authors interpret these changes as (I don’t entirely follow the reasoning) changes not so much in the hydration state of the polymer as in the structure of the hydrogen-bond network, and so speculate that the conformational change involves not just alterations to polymer-water interactions but also water-water interactions.

Time-dependent fluorescence Stokes shifts (TDFSS) are becoming a useful tool to look at water and protein dynamics, the usual approach being to measure the decay of tryptophan fluorescence as a probe of local dynamics. Jay Knutson at NIH and colleagues have used this method to look at relaxation processes in the protein monellin and their coupling to the solvent (J. Xu et al., JPCB 119, 4230; 2015 – paper here). They distinguish two emission processes, which they call genuine and pseudo-TDFSS, and show how to separate them; only the former tells us about the coupling of water and protein dipoles.

One aspect of water’s cell behaviour that is less often discussed is hydrodynamics, which must evidently become important at the mesoscale. Of course, that’s the scale which is very hard to model – but here fluid motions are likely to influence things like protein relaxation and crowding. Fabio Sterpone at the Université Paris Diderot and colleagues present a coarse-grained protein model called OPEP that enables this when combined with a Lattice Boltzmann approach to the fluid kinetics (F. Sterpone et al., J. Chem. Theor. Comput. 11, 1843; 2015 – paper here). They demonstrate its use to look at, e.g. protein transport properties, amyloid aggregation and crowding.

A new approach to modeling water is presented by Vlad Sokhan of the National Physical Laboratory in England and colleagues (V. P. Sokhan et al., PNAS 112, 6341; 2015 – paper here). They say that they can incorporate many-body effects into a coarse-grained parametrization of the electronic structure, which, along with fairly standard point charges and short-range pair potentials, allows accurate prediction of all the bulk behaviour, from liquid-gas coexistence to criticality, freezing and the temperature of maximum density. It’s apparently relatively easy to implement, and they hope to use it to look at effects such as hydrophobic hydration and drying and water’s role in protein association.

More to follow rather soon, I hope.

Monday, April 13, 2015

Hydrophobic or just solvophobic?

As I mentioned in the previous post, the notion of a dewetting transtion – in effect, capillary condensation driven by enhanced density fluctuations – that drives hydrophobic attraction has yet to be fully integrated with the question of whether this is a generic solvophobic effect or something specific to water’s hydrogen-bonded network. David Chandler’s picture of a dewetting transition occurring between extended hydrophobic surfaces for a lateral size scale of around 1 nm or more has tended to focus on the impossibility of maintaining the integrity of the H-bonded network in this geometry. But it may be that the density depletion and enhanced fluctuations on which this picture is predicated are more general features of solvophobicity. Rick Remsing and John Weeks at the University of Maryland speak to this question in a preprint that aims to dissect this hydrophobic interaction into components related to hydrogen bonding and to longer-ranged dispersion and electrostatic forces between the solvent molecules (http://www.arxiv.org/abs/1502.05220). Their conclusions are so nicely summarized in the paper that I can’t do better by paraphrasing them:
“We employ short ranged variants of the SPC/E water model to show that small scale solvation and association in water is governed by the energetics of the hydrogen bond network alone. However when the solute is large and the hydrogen bond network is broken at the hydrophobic interface, water behaves in a manner qualitatively similar to a simple fluid, with unbalanced LJ attractions dominating the solvation behavior.”

For example, without LJ attractions in the solvent, there is no dewetting-induced hydrophobic attraction of two fullerene molecules. (This implies that the crossover between “small” and “large” solutes lies somewhere between the sizes of methane and C60.) In other words, dewetting here is nothing other than regular (albeit barrier-less) capillary evaporation of a solvent, and not a “water effect” at all. Which, if it’s right, means that we might want to think about speaking of a “hydrophobic interaction” at small scales but a “solvophobic interaction” at large scales. But I’d like also to know how this fits with Ronen Zangi’s study indicating that there’s actually a repulsion between fullerenes in water, mentioned in an earlier post. In other words, how potential-dependent is all this?

They’ve been busy. In another contribution, Remsing and Weeks add another variant to the many efforts to develop hydrophobicity scales for biomolecules. This one is based on electrostatics, which has the advantage of being able to predict water-mediated hydrophilic interactions as well as hydrophobic ones (JPCB jp509903n; 2015 – paper here). They begin with a nice description of efforts so far, making the fundamental distinction between “surface-based” methods which aim to use the biomolecular surface properties alone, and “water-based” methods in which the effects of surface topography and neighbouring chemical functionality on the hydrogen-bond network of the local hydration sphere are taken into account. Their new method calls into the latter category, but is computationally inexpensive as it aims to characterize the long-wavelength collective electrostatic response of the water to the surface in question. Not only does this distinguish between hydrophilic and hydrophobic surfaces, but it accounts for different types of hydrophilic surfaces, e.g. those the polarize the water molecules in different orientations. This allows them to identify situations where the approach of two hydrophilic surfaces might induce a water-mediated interaction because of the commensurate polarization of the water network.

That water penentrates into carbon nanotubes, despite its hydrophobic nature, has been confirmed both in simulations and in experiments. In a preprint (http://arxiv.org/abs/1501.0608), Hemant Kumar and colleagues at the Indian Institute of Science in Bangalore ask whether this is driven by entropy or energy. Previous studies have given conflicting answers, but on the basis of MD simulations using the two-phase thermodynamic method Kumar et al. conclude that both energy (the carbon-oxygen LJ interaction) and entropy (for low occupancy, at least) support the filling process. This seems consistent with the findings of J. P. Huang at Fudan University in Shanghai and colleagues that water flows several times faster through non-straight (zigzag) carbon nanotubes than through straight ones, owing to the greater LJ interactions in kinked channels (T. Qiu et al., JPCB 119, 1496; 2015 – paper here).

When a solute particle is excited (electronically, vibrationally, rotationally), how does the (water) solvent relax to the perturbation? Rossend Rey at the Polytechnic University of Catalonia and James Hynes at Colorado have examined this question using linear response theory, and conclude that most of the absorbed energy is transferred to hindered rotations (librations) of the water molecules – and that mostly in the first hydration shell (JPCB jp5113922; 2015 – paper here).

The M2 proton channel of influenza virus A is the target of several flu drugs. These appear to bind to the channel and disrupt the proton transport, which seems to involve water clusters in the channel. By calculating the energetics of pore blockers at different sites in M2, Michael Klein at Temple University and coworkers offer insights into the mechanisms of drug action that might guide the identification of new types of inhibitor (E. Gianti et al., JPCB 119, 1173; 2015 – paper here). The general principle is to dehydrate the pore by replacing the water clusters with the ligand scaffold, and the results here show that this is most effectively done when the ligand scaffold mimics the water-cluster contour, while also preserving the interactions that the cluster made with the protein.

Another water-containing channel is explored by Ai Shinobu and Noam Agmon at the Hebrew University of Jerusalem, and I like their opening line: “Internal water molecules in proteins are conceivably part of the protein structure”. Quite so. They look at the lone water molecule in the “barrel” of the proton-conducting green fluorescent protein, using MD simulations to examine how water exchange occurs following photoexcitation, which opens up the channel transiently (JPCB 119, 3464; 2015 – paper here). The water molecule is shifted by the formation of a water wire through a temporary “hole in the barrel” connecting the chromophore with the bulk: a weak spot between strands of the β-barrel. This wire provides a route for protons to leak out of the channel, and the authors think that this might in fact supply the dominant mechanism for proton escape from the protein. The water motion, meanwhile, involves interactions with hydrogen-bonding residues that result first in sub- and then super-diffusive motion.

How conformationally stable are proteins when dehydrated for storage? One way to find out is to look at water adsorption isotherms as a function of humidity for different conformations. This is what Pablo Debenedetti and coworkers at Princeton have done for the Trp-cage mini-protein using simulations of different protein matrices: crystal, powder, and thermally denatured powder (S. B. Kim et al., JPCB 119, 1847; 2015 – paper here). All three matrices display so-called type II adsorption isotherms, in which there is hysteresis between adsorption and desorption across most of the humidity range. The isotherms are all of similar shape, showing little sensitivity to the degree of ordering in the proteins. Moreover, all show similar changes in swelling behaviour and hydrogen-bonding content as a function of humidity, except for the degree of intra-protein H-bonding, which unsurprisingly depended on the degree of folding in the monomers.

A great deal of attention is now being focused on proteins that have no great degree of conformational regularity in the first place: intrinsically disordered proteins, such as the tau protein which regulates microtubule formation in the nervous system. Dysfuntions in tau can lead to protein aggregation and fibril formation of the sort associated with neurodegenerative diseases such as Alzheimer’s. Martin Weik at Grenoble and coworkers have used neutron scattering and MD simulations to compare the dynamical coupling of protein and solvent through the protein dynamical transition (at 240 K) for the tau IDP and a representative globular protein, the maltose binding protein (G. Schirò et al., Nature Commun. 6, 6490; 2015 – paper here). There is a general notion that the dynamical transition corresponds with the onset of hydration-water translational motion on the protein surface. But although IDPs also show a dynamical transition, they have considerably more solvent-accessible surface than globular proteins, so it is by no means clear that one can expect the same kind of water-protein coupling to apply. But it seems that it does. Martin and colleagues find that in both cases the onset of water translational diffusion seems to coincide with that of large-amplitude protein conformational fluctuations, of the sort needed for functional behaviour. Thus this connection seems to be independent of the protein’s folding state.

What effect do osmolytes such as urea (a denaturant of globular proteins) have on IDPs? That question is explored by Zachary Levine and colleagues at UCSB using a combination of simulations and experiments (PNAS 112, 2758; 2015 – paper here). They find that both urea and trimethylamine N-oxide (TMAO) affect the structure of tau by shifting the distribution of existing conformations rather than by adding any new ones. The osmolytes do so by altering the balance between hydrogen-bonding and salt-bridge interactions in the individual IDPs. In doing so, urea suppresses aggregation, while TMAO promotes the formation of compact oligomers. The mechanism of the latter is subtle, stemming from changes in hydration of the IDP in the presence of TMAO in such a way as to promote aggregation entropically by releasing TMAO and water from the protein surfaces. These predictions of the simulations are borne out by experiments.

Urea can denature RNAs too. Alexander MacKerell at Maryland and colleagues have investigated why (K. Kasavajhala et al., JPCB 119, 3755; 2015 – paper here). The general idea has been that urea forms H-bonds and stacking interactions with the nucleotide bases. That’s a view that is supported by these ab initio calculations, which show that stacking via dispersion forces as well as H-bonding create cage-like complexes around the bases. For example, guanine may become surrounded by 5 urea molecules and 12 waters, pretty decisively trapping it in an unfolded conformation. Direct interactions, you see.

MacKerell also has an interesting paper with E. Prabhu Raman on the energetics of protein-ligand binding (JACS 137, 2608; 2015 – paper here). They have looked in particular at the roles of water in the binding site, and especially the classical view that the “hydrophobic effect” in ligand binding is due to the reduction of nonpolar solvent-exposed area at the binding interface and the concomitant release of more “highly structured” water from this location. To calculate changes in solvation energy as a ligand binds, they use something called Grid Inhomogeneous Solvation Theory (GIST), described by Nguyen et al. in J. Chem. Phys. 137, 044101 (2012). They calculate the various thermodynamic contributions to the binding energy for propane and methanol in several different binding pockets of the proteins Factor Xa and P38 MAP kinase. While they find that the entropy of reorganization of water in the binding pockets favours ligand binding, much as the traditional picture of an entropically driven hydrophobic effect would suggest, the picture is actually rather complex, with subtle interplay between direct protein-ligand interaction energies (even in nonpolar sites) and loss of water interaction energies that can sometimes compensate and lead to a rather small binding enthalpy. Sometimes the enthalpic and entropic changes can oppose (compensate) each other, sometimes they reinforce one another. The general message seems to be that, much as some earlier studies have shown, it is difficult to generalize about the respective contributions to the binding thermodynamics.

More on antifreeze proteins: Aatto Laaksonen and colleagues at Stockholm University show that representatives of the two major classes of AFPs (“hyperactive” and “moderately active”) have different effects on the nature of the ice-water interface when they are bound there (G. Todde et al., JPCB 119, 3407; 2015 – paper here). For the former class, they study the snow flea AFP; for the latter, the winter flounder AFP. Both AFPs increase the thickness of the interfacial region (defined as the region where water diffusion varies from 10 to 90% of the bulk liquid value), with the hyperactive AFP having the greatest effect (widening by 25-40%). This protein has ~25% more hydrophobic surface (the ice-binding side) than the wfAFP, but also 60-70% more hydrophilic surface; the authors think that it’s the first of these differences that counts the most.

The snow flea (a) and winter flounder (b) antifreeze proteins bound at the ice-water interface.

Ariel Fernandez has reported further evidence that the protein regions he calls dehydrons – parts of the backbone where hydrogen bonds in the backbone are “imperfectly wrapped” and thus solvent-exposed – may play not just a structural but also a chemical role (FEBS Letters 589, 967; 2015 – paper here). He has previously argued that water molecules in dehydron regions can act as proton acceptors because of the way that confinement prevents them from orienting their dipoles perfectly with the prevailing electric fields. Now, using quantum calculations, he expands on how this behaviour makes dehydrons activators of nucleophilic groups, and looks at some biochemical consequences, in particular for cancer-related mutations of certain kinases. The nucleophilicity induced by the dehydron, he says, turns the kinase constitutively (and hazardously) active for phosphorylation.