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.

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.

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.

JCP special issue on biological water

The main news this time round must be the “special topic” issue of J. Chem. Phys. on “biological water”, edited by Gerhard Hummer and Andrei Tokmakoff (here; see their preface here). I’m not going to go through all the contributions, nor invidiously to pick out any ones in particular – needless to say, it’s a treasure trove, and well worth browsing.

A completely new motif for ice binding in an antifreeze protein has been identified by Peter Davies and colleagues at Queen’s University in Kingston, Ontario, in a midge native to Lake Ontario (K. Basu et al., PNAS 112, 737; 2015 – paper here). It is a small protein of just 79 residues, and contains a 10-residue coil crosslinked by a disulfide bond, which presents seven tyrosine chains in a flat array for ice binding. The protein, which has no known homologues, seems to be a relatively “cheap” means of protecting the midges from occasional night frosts when they emerge in the spring.



The idea that protein denaturants such as guanidinium cations and urea act via direct interactions with the protein is explored by Sandeep Patel and colleagues at the University of Delaware, who use MD simulations to look at the orientations of these molecules close to hydrophobic residues and how these affect solvent fluctuations in the vicinity (D. Cui et al., JPCB 119, 164; 2015 – paper here). They find a correlation between the stability of the molecular arrangement at the interface (e.g. parallel or perpendicular orientations of the denaturant) and the degree of fluctuation induced – implying that the effects of the denaturants may be related to their tendency to introduce malleability in the local hydration shell, with consequences for hydrophobic interactions.

Intracellular lipid-binding proteins (iLBPs) are central to fatty-acid uptake, transport and metabolism. They have an intriguing structure, in which a β-barrel contains a large water cluster. Shigeru Matsuoka and colleagues at Osaka University have attempted to figure out what the role of these waters is, using MS simulations of human-heart-type fatty-acid binding protein (FABP3), a typical iLBP (D. Matsuoka et al., JPCB 119, 114; 2015 – paper here). They observe two conformations. The empty cavity is wide open, with a relatively ordered hydrogen-bond network inside it. But when the ligand is bound, some of the waters exit through a “back portal” while others provide a hydrogen-bonded latch, connecting to highly conserved hydrophilic residues, that “seals” the cavity with the fatty acid (stabilized by hydrophobic interactions with the interior) inside.

In an arxiv preprint (1412.2698 – paper here), Martin Wolf and colleagues at Augsburg present dielectric measurements of hydration water dynamics of lysozyme in frozen solution, ordinary solution and a hydrated powder. They claim to find clear evidence for bimodal dynamics, with a crossover that supports the notion of a fragile-to-strong transition in the metastable “No Man’s Land” regime.

(Incidentally, did I detect in Twitter some skepticism about how so many of these studies present their results as N=1 samples consisting of lysozyme or HIV protease? If so, it’s a fair concern, but you have to start somewhere. As Wolf and colleagues acknowledge, further studies are needed here to determine the generality.)

The fact that I write every month for Nature Materials makes it all the more unforgiveable that I failed to spot the paper there by Wang et al. in 2013 showing how water may play a vital structural role in bone apatite (Nat. Mater. 12, 1144 – paper here). By way of atonement, let me point to a nice contribution by Neeraj Sinha at the Center of Biomedical Research in Lucknow and colleages who describe a solid-state NMR method for investigating the nature of collagen hydration in the bone matrix (R. K. Rai et al., JPCB 119, 201; 2015 – paper here). They say that the hydration in situ seems to be significantly different from that of extracted collagen, and adds to the idea that water plays a big part in determining and modifying the mechanical properties.

We need new flu drugs, agreed? Bill DeGrado at UCSF and colleagues report a new strategy for designing inhibitors of flu viruses that bind in two variants of the M2 proton channels (WT and S31N), where a network of water molecules assists proton transport (Y. Wu et al., JACS 136, 17987; 2014 – paper here). For these two strains, in which the inhibitor binds with different orientations, the initial results suggest that the antiviral activity should be comparable to the existing antiviral amantadine.



The role of hydration waters in the sequence-specific bending flexibility and conformational stability of DNA is studied in a paper by Jerzy Leszczynski at Jackson State University and colleagues (T. Zubatiuk et al., JPCB 10.1021/jp5075225 – paper here). They look specifically at A-tracts of B-DNA, which resist deformations and so might be important in the organization of nucleosomes. They find that this stiffening is influenced by the specific patterns of structural water within both the major and minor grooves

Tunneling and delocalization of protons in a hydrogen-bonded network in the active site of ketosteroid isomerase is described in ab initio simulations by Thomas Markland and colleagues at Stanford (PNAS 111, 18454; 2014 – paper here). They say that this leads to a boost in the acidity of the site by around four orders of magnitude compared with the classical situation. There are no actual waters in the site in this case, but the general implication seems clear enough.



Another nice example of “functional water” at an active site: Vivek Sharma at the Tampere University of Technology in Finland and colleagues say that an organized cluster of water molecules in cytochrome c oxidase helps to steer proton transfer in the right direction, so that the electron transfer in the reduction of oxygen to water can be coupled to proton pumping across the membrane (PNAS 112, 2040; 2015 – paper here). Their simulations indicate that the waters reorganize in a redox-dependent way so as to coordinate electron transfer with proton transfer along the requisite path (see diagram of the two states below).



There is increasing reason to believe that hydrophobicity is a context-dependent property. That conclusion might be read into the findings of Dor Ben-Amotz and colleagues at Purdue on how the presence of a charged group such as carboxylate or tetraalkylammonium alters the hydration structure around an adjacent aliphatic chain (J. G. Davis et al., JPCB jp510641a – paper here). They found in previous work (Nature 491, 582; 2012) there seems to be a change in the hydration structure of aliphatic alcohols at a chain length of around 1 nm, consistent with the proposal of such a crossover by David Chandler and colleagues. Using Raman spectroscopy and simulations, they now see a similar structural change for aliphatic carboxylic acids with different chain lengths, but that this transition is suppressed when the carboxylic acids are deprotonated. The effect is stronger for tetraalkylammonium ions – that is, the perturbation created by the charged head group extends several methylene groups down the aliphatic tail. So it would seem that these charged head groups alter the hydrophobic hydration in ways that neutral hydrophilic groups do not.

This fits very nicely with recent work by Sam Gellman, Nicholas Abbott and colleagues at Wisconsin-Madison, who find that the hydrophobic interactions of methyl-terminated self-assembled monolayers, as measured by chemical force microscopy and single-molecule force measurements for docking of the nonpolar domains of peptides, are altered by the inclusion of some cationic amine and guanidinium groups among the SAM (C. D. Ma et al., Nature 517, 347; 2015 – paper here). Curiously, protonated amine groups enhanced the hydrophobic interactions, while charged guanidinium groups suppress them. Gellman and colleagues reach much the same conclusion as Dor’s team: that the results are consistent with the idea that “solvation shells are susceptible to the influence of neighbouring atoms, especially strongly charged ones.” There’s a nice News and Views piece accompanying the paper by Shekhar Garde (Nature 517, 277 – here).

How does dewetting between two hydrophobic surfaces happen? The classic picture is that this is capillary condensation, a first-order process that requires the formation of a nucleus of a critical size – say, a vapour tube bridging two planar hydrophobic surfaces. But Richard Remsing at the University of Pennsylvania and coworkers report simulations in which dewetting seems to circumvent this classical mechanism: fluctuations of the confined water give rise to a cavity larger than the size of a critical nucleus, which then grows spontaneously (R. Remsing et al., arxiv 1502.05436 – paper here). This certainly fits with earlier suggestions by the Chandler group that dewetting is driven by solvent fluctuations.

I wonder, though, if we have an adequate picture of how these fluctuations fit within a standard picture of confined fluids. Where do they come from, and are they specific to water or a consequence of solvophobicity more generally? I have been talking to (admission of interest: my one-time PhD supervisor) Bob Evans at Bristol about this, who pointed me to a paper in press with J. Phys. Cond. Matt. with Maria Stewart which hopes to shed light on the issue. They point out that even for simple Lennard-Jones fluids close to solvophobic walls there is an enhancement, over just a molecular diameter or two, of the local compressibility. This, they say, looks very much like the “enhanced fluctuations” that David Chandler, Shekhar Garge and others have reported for water at hydrophobic surfaces. In other words, this local compressibility, analogous to the surface susceptibility in an Ising model that serves as an indicator of the approach to a critical wetting or drying transition – offers a properly defined thermodynamic measure of what previously has been a rather vague, hand-waving concept. And it implies that there is nothing here that is unique to water.

How much do quantum effects that allow for electronic polarization matter to the energetics of ligand-receptor binding calculations, relative to molecular mechanics calculations that neglect them? Adrian Mulholland and colleagues at Bristol address this question by looking at the binding of water molecules within the cavity of the influenza viral protein neuraminidase, a target for antivirals such as oseltamivir and peramivir (C. J. Woods et al., JPCB 119, 997; 2015 – paper here). They consider this system because of the recognized importance of bound water molecules in drug design, and in particular the known function of a water molecule in oseltamivir binding. Structural studies of ligand-neuraminidase complexes have revealed two kinds of important “structural” waters: one in a hydrophilic site, the other a hydrophobic one. They find that in going from molecular modeling to ab initio calculations, the difference in binding energy due to water polarization can be as much as 1 kcal/mol, and is greater for the hydrophilic water.

Laurence Pratt at Tulane University and his coworkers have dissected the role of intermolecular forces on hydrophobic interactions (M. I. Chaudhar et al., arxiv preprint 1501.02495 – paper here). They find that for hydrated argon atoms, including realistic solute attractive forces substantially weakens the hydrophobic attractions relative to the situation without them (as assumed in Pratt-Chandler theory formulated in 1977 for hard spheres: JCP 67, 3683). Among other things, it’s pretty remarkable that Laurence has returned to reconsider the idea after almost 40 years!

This is not directly about hydration, but it’s involved: I have a story here in Chemistry World about a new computational method for calculating the free energies of ligand binding to a wide range of protein receptors (L. Wang et al., JACS 137, 2695; 2015 – paper here). The authors claim that it comes within 1 kcal/mol of the measured binding energies for most of the cases studied. As far as hydration is concerned, Robert Abel at Schrodinger, the company that developed the method, says to me that “These simulations use explicitly solvated molecular dynamics. So, molecular hydration affects should be well-described. Such an example of a ligand displacing a water from a cavity is described in in figure 4 sub-panel C and at the bottom second column of page 2699 of the manuscript.”