Water and protein folding

Does water drive protein folding? That’s the title of a paper by Yutaka Maruyama and Yuichi Harano at Osaka University (Chem. Phys. Lett. 581, 85; 2013 – paper here), and as you’d expect from a title like that, they conclude that the answer is at least partly affirmative. They combine MD simulations and 3D-RISM solvation theory to calculate thermodynamic quantities involved in protein folding, and conclude that while hydration energy favours the denatured state (but is largely offset by the intramolecular interactions of the protein on folding), hydration entropy favours the folded state. In this sense, water contributes significantly to the stability of the collapsed conformation.

A perhaps more dramatic claim along these lines is made by Ariel Fernandez, now at the Argentinian Institute of Mathematics in Buenos Aires (J. Chem. Phys. 139, 085101; 2013 – paper here). He presents a model of electrostatic interactions between the protein surface and water dipoles, which suggests a principle that Ariel calls minimal episteric distortion: the protein-water interface adopts a configuration that minimizes the energetic cost of disrupting dipole interactions of the hydrogen-bonded matrix. In other words, the native fold is the one that corresponds to this minimally perturbing topology: the protein structure is the one that is least at odds with the structure of the surrounding water. As Ariel has put it, “The paper shows that the full interfacial free energy can be computed/interpreted in two different but equivalent ways: a) as elastic energy arising from the perturbation of the hydrogen-bond matrix of water, or b) as electrostatic energy stored in the anomalous polarization of water (interactions between dipoles forced not to behave in Debye manner and protein charges).” And crucially, if this interfacial free energy is omitted from the folding computations, an incorrect fold results because the structure is then “at odds with the solvent”.

John Weeks at the University of Maryland was one of the architects of the Lum-Chandler-Weeks hypothesis (J. Phys. Chem. B 103, 4570; 1999) that the hydration of small and large hydrophobes is qualitatively different, with a crossover scale of around 1 nm, and that the latter is dominated by interfacial free energies and the breaking of hydrogen bonds. (This was itself an extension of a proposal by Frank Stillinger in 1973.) Now Weeks, together with Richard Remsing at Maryland, has attempted to refine this idea by considering what roles van der Walls dispersion forces and electrostatic interactions might have in this business. Using MD simulations with the SPC/E water model, they confirm the original idea that solvation and association of small hydrophobes is dominated by hydrogen-bonding, but find that for large hydrophobes (where hydrogen bonds are broken at the interface) the attractions are dominated by Lennard-Jones dispersion forces, with water now behaving more like an ordinary liquid (J. Phys. Chem. B jp4053067 – paper here). The crossover length scale is set by the hydrogen-bond network itself, occurring when the solute is too large for this network to remain intact. This paper seems to offer an important extension of LCW/Stillinger, while showing that the basic ideas expressed in those earlier works are sound.

Further support for this picture comes from Guillaume Jeanmairet and colleagues at the École Normale Supérieure in Paris. They have recently presented a now molecular density-functional theory of water (J. Phys. Chem. Lett. 4, 619; 2013). In a preprint, they have now applied it to the hydration of hydrophobes of various sizes (G. Jeanmairet et al., arxiv.org/abs/1307.7237). They find that the original theory doesn’t work so well, in terms of predicting solvation free energies, when applied to large hydrophobes, but that supplementing it by introducing a hard-sphere component that reproduces the van der Waals picture of liquid-vapour coexistence in bulk rescues the model. This seems to be, they say, very much in line with the Lum-Chandler-Weeks picture of a crossover between “volume-driven” hydrophobic hydration at small scales and a surface-driven picture, dominated by interfacial free energies, at larger scales.

Rahul Sarma and Sandip Paul of the Indian Institute of Technology in Guwahati Assam have been rather extensively investigating the origins of osmolyte effects on protein stability using MD simulations. In their latest paper (J. Phys. Chem. B 117, 9056; 2013 – paper here) they look at the stabilizing effect of trimethylamine-N-oxide (TMAO) against the pressure-denaturation of proteins. They find that TMAO enhances the hydration number of a coiled peptide more than its extended state under pressure, whereas the opposite is seen in its absence. Partly this is an indirect effect: solvation of TMAO reduces the ‘crowding’ of water molecules at high pressure that drives them towards the relative freedom they find in the hydration shell of the extended peptide. There is also a direct effect due to the relative inefficiency with which TMAO interacts with the unfolded peptide.

Alkaline phosphatase enzymes are able to catalyse the hydrolysis of a range of different phosphate and sulphate substrates. What accounts for this promiscuity? Through first-principles simulations, Guanhua Hou and Qiang Cui at the University of Wisconsin-Madison show that this class of enzymes can support different types of transition state in the same active site (JACS 135, 10457; 2013 - paper here). The differential placement of water molecules in the active sites is implicated in some of this, although the details are complicated and apparently as yet case-specific: there is no general picture emerging yet of how these and other enzymes engineer their promiscuity.

Another water molecule playing a functional role in enzyme catalysis is identified by Walter Thiel and colleagues at the MPI für Kohlenforschung in Mülheim (B. Karasulu et al., JACS 135, 13400; 2013 – paper here). They look at the three-molecule water bridge that exists in the active site of a lysine-specific demethylase (which is involved in demethylation of histones), showing how it is stabilized by interactions with the surrounding residues and how it assists proton transfer.



Electrowetting – switching surfaces between hydrophobicity and hydrophilicity with electric fields – has useful applications in microfluidics and ink-jet printing, and might enable voltage-gating of flow through nanochannels. This provides the motivation for a study by Alenka Luzar at Virginia Commonwealth University and her colleagues of the effect of electric fields on water dynamics at the interfaces of a slit-like nanopore (M. von Domaros et al., J. Phys. Chem. C 117, 4561; 2013 – paper here). They recognize that the application of a field across the pore breaks the symmetry so that the water dipoles tend to be aligned in opposite directions with respect to the surfaces at each interface – this becomes, they say, the electrical equivalent of a chemical Janus interface. Their simulations indicate that the water density in the first hydration layer is lower for the “incoming” than for the “outgoing” field, and that the response times for orientational dynamics can be up to two orders of magnitude different (in the picosecond regime) – an effect that ought to be visible to, for example, dielectric spectroscopy.

Pavel Jungwirth, Damien Laage and their colleagues have looked at how hydrated ions affect water dynamics (G. Stirnemann et al., JACS 135, 11824; 2013 – paper here). Their simulations indicate that in dilute solution, effects on water reorientational dynamics are short-ranged and ion-specific: both retardation and acceleration may be seen, depending on the interaction strengths. But in concentrated solution, where hydration shells overlap, the effect is always a retardation and is not ion-specific. They suggest that this might explain the apparent discrepancy between the studies of water dynamics by NMR (at low salt concentration) and by infrared and THz spectroscopy (e.g. Huib Bakker), which must use high salt concentrations.

Niharendu Choudhury and colleagues at the Bhabha Atomic Research Centre in Mumbai offer a new view of the local solvation environment in pure water (J. Phys. Chem. B 117, 8831; 2013 – paper here). They say that, by considering the hydration shell of each molecule to be composed only of other waters hydrogen-bonded to it, they can explain the density anomaly without recourse to polyamorphism of the liquid state. I confess that I’ll need to digest this paper more to see where it really diverges from previous treatments; in essence it seems to posit three order parameters, namely a tetrahedral order parameter in the first hydration shell, an orientational order parameter in the second shell, and the number of hydrogen bonds. I think the point is that the first two can be subsumed in the third, and that distortions to the tetrahedral coordination arise at higher temperatures from the incursion of non-hydrogen-bonded molecules into the first shell. The resulting analysis restores a picture of water structure in terms of fluctuations in a homogeneous fluid, rather than requiring the kind of heterogeneities posited by Anders Nilsson.

Ali Hassanali at ETH and coworkers have uncovered what seems to be an extraordinary complexity and subtlety in motion of protons through water (A. Hassanali et al., PNAS 110, 13723; 2013 – paper here). (See also commentary by Edelsys Codorniu-Hernández and Peter Kusalik of the University of Calgary here.) Their first-principles simulations show that there is a great deal more to it than Grotthuss hopping. For a start, the process is very heterogeneous in time: both proton and hydroxide diffusion happen in bursts of activity followed by periods of quiescence. These motions involve medium-range correlations between the movements of many protons, which the authors characterize in terms of the connected-ring structure of the hydrogen-bonded network. The result is that protons can jump over a wide range of length scales, and not merely hope from one molecule to the next. The authors point out that “the fundamental aspects raised in this re- port are likely to open up new directions in the role of the water network in phenomena associated with the hydration of ions and macromolecules such as proteins and DNA.”

Making water modelling easier: Vijay Pande at Stanford and colleagues have simplified the AMOEBA polarisable model of water to a version they call inexpensive or iAMOEBA, which introduces a direct polarization approximation that removes the need to calculate polarization iteratively (L.-P. Wang et al., J. Phys. Chem. B 117, 9956; 2013 – paper here). Any shortfall in the polarization energy introduced by the approximation can be recovered by parametrization, so that the model accurately reproduces the water/ice phase diagram, dielectric properties and so forth.

Spectral tuning and pumping in rhodopsins

Inert gases in water will under some conditions not exhibit the expected hydrophobic attraction but will form a solvent-separated pair. Yuri Djikaev and Eli Ruckenstein at SUNY at Buffalo rationalize this observation in the course of developing a new approach to describing hydrophobic hydration (J. Phys. Chem. B 117, 7015; 2013 – paper here). They derive an analytical expression for the number of hydrogen bonds each water molecule has as a function of its distance from the hydrophobic particle, and find that the hydration energy can be positive even for apolar particles if they are small enough, thanks to van der Waals interactions.

On the other hand, two guanidinium ions can form a like-charge ion pair in water, as reported by Richard Saykally and colleagues at Berkeley from XAS measurements (O. Shih et al., J. Chem. Phys. 139, 035104; 2013 – paper here). Their calculations indicate that the ions form a stacked arrangement through pi* interactions.

It is very nice to see that Bruce Berne and colleagues are extending the work on drying transitions in protein assembly to their potential role in molecular recognition and ligand binding (J. Mondal et al., PNAS 110, 13277; 2013 – paper here). Their simulations of an idealized hydrophobic particle being bound in a cavity indicate that the latter can fluctuate between wet and dry states, which have distinct kinetic binding profiles.

More on water’s liquid-liquid transition: Yaping Li and colleagues at the University of Arkansas see such a first-order transition between HDL and LDL, with an associated critical point, in simulations using the so-called Water potential from Adaptive Force Matching for Ice and Liquid (WAIL), which they argue is a particularly reliable potential because it is not biased by fitting to experimental data (Y. Li et al., PNAS 110, 12209; 2013 – paper here).

And in a preprint apparently heading for the Eur. Phys. J., Hajime Tanaka presents a new model of the liquid state based on an order parameter that describes bond orientational ordering, which he says explains many of the anomalies of tetrahedrally bonded liquids (including a liquid-liquid transition) (arxiv.org/abs/1307.0621). This huge and ambitious paper also provides accounts of glass formation and fragility, quasicrystal formation and other metastable crystallized states.

Somewhat related in exploring tetrahedral order in water is a preprint destined for Chem. Phys. Lett. by Marcelo Carignano of Northwestern University and colleagues, who have compared this aspect of water structure along with hydrogen-bond lifetimes and diffusion coefficients in simulations with four different water potentials (SPC/E, TIP4P-Ew, TIP5P-Ew, Six-site) (arxiv/1307.3611). They find that there are significant differences below 270 K, depending on whether or not the models include explicit lone pairs.

Shaoyi Jiang and colleagues at the University of Washington in Seattle present a case, from simulations and NMR measurements, that the hydration differences for salt bridges in proteins contribute significantly to their relative stabilities (A. D. White et al., J. Phys. Chem. B 117, 7254; 2013 – paper here).

More on spectral tuning of photoactive sites by hydration environments. Sivakumar Sekharan and colleagues at Yale say that the chloride-pumping transmembrane retinal protein halorhodopsin in a halophile works by subtle rearrangements of waters and ions in the vicinity of the chromophore as chloride translocation progresses, inducing changes in chromophore bond lengths that affect its absorption wavelength (R. Pal et al., JACS 135, 9624; 2013 – paper here). Meanwhile, Hyun Woo Kim and Young Min Rhee at the Pohang University of Science and Technology in Korea say that the pH-dependence of emission from firefly oxyluciferin – a redshift in acidic conditions – seems to be fine-tuned by a water molecule in the active site that can mediate the dynamics of neighbouring groups to the chromophore (J. Phys. Chem. B 117, 7260; 2013 – paper here).

Valentin Gordeliy of the Université Grenoble Alpes and colleagues describe the mechanism of a different rhodopsin-like pump, an unusual proteorhodopsin from a permafrost bacterium that pumps protons in a different manner to other known rhodopsins (I. Gushchin et al., PNAS 110, 12631; 2013 – paper here). Unusually, this structure has a proton release site (a lysine) already connected to the bulk solvent by a water chain in the ground state. There’s a nice graphic that illustrates the similarities and differences in the water-containing cavities of the channel for this structure and other rhodopsins, bacteriorhodopsin and xanthorhodopsin, the closest homologue of this one (ESR):



I guess it is ultimately a cautionary tale that one should extract from the simulation study of reverse micelles by John Straub and colleagues at Boston University: they say that the structure and dynamics of the encapsulated water and the overall micelle assembly depend strongly on the force fields used (A. V. Martinez et al., J. Phys. Chem. B 117, 7345; 2013 – paper here). In any event, the micelles tend not to be spherical but undergo pronounced shape fluctuations, a point made also by earlier simulations but which tends to be ignored in interpreting experimental data.

I must confess that I’d not previously come across inhomogeneous fluid solvation theory (IFST) as a general method for calculating the effect of a solute on the solvent structure. But now David Huggins and Mike Payne at Cambridge have assessed how well it does for predicting the hydration free energies of small solutes (J. Phys. Chem. B 117, 8232; 2013 – paper here). They look at six solutes such as benzene, isobutene and methanol, all in water, and figure that IFST does pretty well in comparison with a more sophisticated theory but that accurate prediction of entropies might suffer from poor sampling of the available configurations unless the simulation times are rather long.

And here’s a curious one: George Reiter at Houston and colleagues say that water confined on the scale of about 2 nm has a different ground-state configuration of valence electrons than in the bulk (G. F. Reiter et al., Phys. Rev. Lett. 111, 036803; 2013 – paper here). They explore the issue using X-ray Compton scattering to probe water in the pores of Nafion, but they say, reasonably enough, that “Biological cell function must make use of the properties of this [nanoconfined] state and cannot be expected to be described correctly by empirical models based on the weakly interacting molecules model.”

A role for water in allostery

Rearrangement of the water network hydrating a protein can provide a mechanism for allostery, according to a study by Peter Hamm and colleagues (B. Buchli et al., PNAS 10.1073/pnas.1306323110; paper here). They insert an azobenzene photoswitch in the binding groove of a PDZ domain protein, a common system for studying allostery, such that photo-induced isomerisation induces a conformational change similar to that which occurs on ligand binding. This enables them to use fast IR spectroscopy to look at the opening of the binding groove in a precisely controlled fashion, having first characterized the equilibrium structures using NMR. Their MD simulations show that a change in water density in the vicinity of the photoswitch right after switching propagates slowly through the water network over about 100 ns until it reaches the back of the protein. They suggest that this change in hydration structure could then induce, either dynamically or structurally, a remote allosteric change in protein conformation. In such a case, this would be a particularly dramatic example of how the hydration network is really a part of the functional apparatus of the protein.

How aquaporins transport water across membranes – and specifically how they do so without also transporting protons – has been a topic of much debate. The consensus has come to focus on the so-called NPA motif in the centre of the channel, a bottleneck through which water kolecules pass in single file, which seems to prohibit proton transport via electrostatic repulsion. Urszula Kosinska Eriksson at the University of Gothenburg and colleagues have recently reported a new high-resolution crystal structure of yeast aquaporin 1 which sheds new light on the issue (Science 340, 1346; 2013 – paper here). As Jeff Abramson and Armand Vartanian of ULCA explain in an accompanying perspective (Science 340, 1294 – paper here), the structure shows that proton transport isn’t (as some have suggested) blocked by hydrogen-bonding of a single water molecule in the NPA region to two asparagines. Rather, there are two independent waters here, but the interactions with the asparagines constrain their dynamics in such a way as to effectively break the ‘water wire’ threading through the channel. The authors say that water transport then seems to happen in pairwise fashion, similar to the ion transport in potassium channels – a possible example of convergent evolution to solve related problems.

A carefully structured water cluster also seems to play an important role in oxygen evolution during the photocycle of photosystem II, as Brandon Polander and Bridgette Barry of Georgia Tech deduce using laser flashes to induce the process and following it by FTIR spectroscopy (PNAS 10.1073/pnas1306532110 – paper here). The hydrogen-bonded cluster of five water molecules, bound to the catalytic Mn4CaO5 cluster, seems to become protonated during the S1→S2 part of the cycle, and it stores the proton until a later stage of the reaction. Ammonia can poison the reaction by disrupting the water cluster.

Water molecules that gain access to the interiors of globular proteins can act as probes of the intrinsic conformational dynamics that enable proteins to function. These waters in the ‘dry’ protein interior have been studied by magnetic relaxation dispersion spectroscopy, but atomistic models are needed to interpret those results. The problem is that such deep water penetration tends to be a rare event, demanding very long (millisecond) run times for simulations. A technique has recently been developed that enables this (D. E. Shaw et al., Science 330, 341; 2010), and now Filip Persson and Bertil Halle at Lund have used the method to compare MD with the MRD experiments for bovine pancreatic trypsin inhibitor (JACS 135, 8735; 2013 – paper here). They find that some of these internal hydration sites have water residence times of several microseconds, and that the water molecules gain access along single-file hydrogen-bonded chains.

Dewetting transtions are known to be important for at least some instances of hydrophobic assembly, and Bruce Berne and colleagues at Columbia now extend their earlier studies of dewetting-induced protein collapse to look at the potential role of such transitions in the docking of hydrophobic ligands in their binding pockets (J. Mondal et al., preprint http://www.arxiv.org/abs/1305.7505). In this way the solvent dynamics, which are retarded in the concave cavity, are explicitly included in the kinetics of the binding process – the process can be parametrized through a state variable that describes whether the pocket is ‘wet’ or ‘dry’, while the ligand diffuses across a potential-energy surface that can switch between these two states.

Daniel Sindhikara and Fumio Hirata of the Ritsumeikan University in Japan present a fast algorithm, based on the three-dimensional reference interaction site model (3D-RISM), for calculating the solvent distribution around solutes (J. Phys. Chem. B 117, 6718; 2013 – paper here). This is a wholly theoretical approach derived from the Ornstein-Zernicke equation. They say that it gives water positions and orientations that agree well with available experimental data, and demonstrate its use on HIV-1 protease.

Paul Ben Ishai at the Hebrew University of Jerusalem and colleagues have used quasielastic neutron scattering to look at salt effects on water dynamics (J. Phys. Chem. B 117, 7724; 2013 – paper here). They find that water diffusion is slower than in pure water, on average, in NaCl solution, but faster in KCl. They interpret the result in terms of structure-making and –breaking, saying that the disruption of the hydrogen-bonding network by potassium ions accounts for its apparent ‘lubricating’ effect.

The hydration state of arginine side chains can be deduced from its UV resonance-enhanced Raman spectrum, according to Sanford Asher at Pittsburgh and colleagues (Z. Hong et al., J. Phys. Chem. B 117, 7145; 2013 – paper here). Their density-functional calculations show that a particular vibration of this residue is sensitive to hydration. They use this signal to characterize differing degrees of hydration of Arg in two polyAla model peptides.

Complexity of osmolytes

More on the mechanisms of denaturation and osmolyte stabilization. Trimethylamine-N-oxide (TMAO) is known to confer protection against protein denaturation by urea. Rahul Sarma and Sandip Paul at the Indian Institute of Technology have carried out MD simulations to try to figure out why (J. Phys. Chem. B 117, 5691; 2013 – paper here). They say that TMAO interacts with a model peptide, N-methylacetamide (NMA), so as to cause some dehydration by replacing solvation water. However, this interaction with the peptide is relatively inefficient, especially because TMAO cannot donate its hydrogen to the backbone carbonyls. As a result, TMAO is not efficient at stabilizing the unfolded peptide chain. If the protein folds, then TMAO is more available for forming very strong hydrogen bonds with water, and indeed with urea. It’s a subtle argument in which the direct interactions between all four components – TMAO, urea, peptide and water – are implicated.

Much the same issues are explored in a new preprint by Bruce Berne and coworkers at Columbia (J. Mondal et al., http://www.arxiv.org/abs/1306.4642). They look at the interactions of urea and TMAO separately with a model 32-mer made of Lennard-Jones beads whose hydrophobicity can be varied. Although both small solutes interact strongly with the polymer, TMAO destabilizes extended conformations while urea stabilizes them. This seems to be because TMAO molecules are more stable (via van der Waals interactions) next to the collapsed rather than the extended polymer. The results provide further support for an interpretation of osmolyte effects that invokes direct interactions rather than indirect effects on “water structure”.

The subtlety of mixed-osmolyte solutions is revealed in simulations of a hydrophobic polymer in urea and guanidinium chloride by Payel Das at IBM Yorktown Heights and colleagues (P. Das et al., Langmuir 29, 4877; 2013 – paper here). They find that, while both of these molecules act as denaturants on their own, in combination they actually promote collapse of the polymer. It is hard to tell a simple story about why this happens, although apparently Paul Flory predicted back in 1955 that two good solvents could combine to induce polymer chain collapse. Although Gdm has the stronger interaction with the polymer, its enhanced concentration in the vicinity of the polymer attracts urea due to the favourable urea-Gdm interaction, with the result that urea is in fact preferentially adsorbed onto the polymer. This sets up a long-ranged interaction between the monomers mediated by their clouds of urea molecules, ultimately driving collapse.

The effect of progressive dehydration of a protein (lysozyme) on its structure and dynamics is studied using Raman spectroscopy by Gediminas Niaura of Vilnius University of colleagues (J. Phys. Chem. B 117, 4981; 2013 – paper here). They find that there is a structural change in the dry protein crystal that begins at about 7-10 wt% water, and that the native state is reached at about 35 wt% water (which amounts to appreciably more than monolayer coverage). In the dry state the protein is dynamically glassy, with a diminished proportion of alpha helices and an enhanced content of beta-sheet contacts.

And on the question of ‘dry’ proteins, Eric Gloaguen at the CNRS Laboratoire Francis Perrin in Gif-sur-Yvette and colleagues report that small peptides with aromatic residues will fold into hydrophobic domains even in the solvent-free gas phase, showing that this is a favourable conformation even in the earliest stages of protein folding (E. Gloaguen et al., J. Phys. Chem. B 117, 4945; 2013 – paper here).

Somedatta Pal and Sanjoy Bandyopadhyay at the Indian Institute of Technology in Kharagpur have used MD simulations to look at the complementary issue of how protein (here barstar) dynamics affect the dynamics of hydration water (J. Phys. Chem. B 117, 5848; 2013 – paper here). They compare the normal situation of the hydrated protein with that in which the protein is kept frozen, looking specifically at the effect of the change on the low-frequency vibrations of the water molecules. Freezing the protein results in stronger confinement of water bound to the protein surface, with a corresponding blue shift of the vibrational frequency for transverse water oscillations – but much less effect on longitudinal oscillations.

An intriguing paper by Nicholas Spencer and colleagues at ETH investigates hydration forces for glycoproteins using the surface force apparatus (R. M. Espinosa-Marzel et al., Biophys. J. 104, 2686; 2013 – paper here). They attach these onto hydrophobic and hydrophobic surfaces, and find that there is a rather long-ranged repulsive force (several tens of nanometres) between the surfaces. It is good to see this neglected class of biological molecules get some attention in terms of their fundamental hydration characteristics. However, I don’t buy the interpretation that the repulsion is due to some “long-ranged structuring of water”. The whole discussion around the results seems to be something of a throwback, starting with the whole notion of “vicinal water” and involving two-state water theories, kosmotropes and all the rest of the paraphernalia that tended to surround discussions of ‘water structure’ 20 years ago. “Ordering” of water over 30 nm or so just isn’t any longer consistent with what is known of other systems, and it’s been suggested to me that steric repulsion of the surfaces due to a few of the macromolecules protruding from the monolayer is a much more likely explanation of the effects observed.

But could it be that long-ranged water structuring is going to try to stage a little comeback? I ask this because it is invoked in another recent paper too, by Jan Christer Eriksson and Ulf Henriksson of the Royal Institute of Technology in Sweden (Langmuir 29, 4789; 2013 – paper here). They develop their earlier argument (Langmuir 23, 1126; 2007) that the long-ranged hydrophobic attraction might be accounted for by the formation of roughly cylindrical bridging water clusters that are “slightly more organized than the rest of the film”. Their analysis suggests that such a proposal can explain some recent measurements on water thin films with the SFA (Wang et al., J. Coll. Int. Sci. 364, 257; 2011). But I think I will stick with the bridging-nanobubbles idea.

No ice please

I’m not a fan of the Frank-Evans iceberg picture of hydrophobic hydration. But I will happily admit that, on the question of whether water hydrating small nonpolar solutes is more/less ordered and mobile than that in the bulk, the evidence is mixed. Nuno Galamba of the University of Lisbon now offers some more support for a weak version of the iceberg picture (J. Phys. Chem. B 117, 2153; 2013 – paper here). His MD simulations show that a subset of water molecules in the first hydration shell of methane have “significantly enhanced tetrahedrality and a slightly larger number of hydrogen bonds”, as well as slower reorientational dynamics, relative to the bulk. He adds that these characteristics should not be visible in the rdfs deduced from neutron scattering, explaining why they have not been seen experimentally. Whether this view extends to large hydrophobes is another matter, but these results at least argue for some small degree of water ‘ordering’, even if this is very far from ice-like.

The role of hydration water in the dynamics of hydrophobic sidechains of peptides is explored by Daniela Russo at the ILL in Grenoble and colleagues, using inelastic neutron scattering and simulations (J. Phys. Chem. B 117, 2829; 2013 – paper here). They say that the activation energy for methyl group motions increases with increasing level of hydration but eventually reaches a plateau when an extended hydrogen-bonding network is established around the group, which happens when it is essentially surrounded by a single layer of water. These sidechain dynamics seem to have a critical impact on the flexibility of the peptide as a whole, and so the degree of hydration appears to determine the onset of conformational freedom for the entire polypeptide.

Neeraj Sinha and colleagues at the Centre of Biomedical Magnetic Resonance in Lucknow have taken on the challenge of exploring water-protein interactions in a rather complex system, the helical coat protein of the filamentous virus Pf1 (R. N. Purusottam et al., J. Phys. Chem. B 117, 2837; 2013 – paper here). Using 2D proton-N15 NMR, they deduce that the filamentous assembly has a highly hydrated core which not only acts as a ‘glue’ but might also mediate the interaction of the arg44 residue with DNA.

Membrane transporters are membrane protein channels that pump directional transport of small molecules across the membrane, coupled to chemical energy sources in the cell. Their operation has generally been considered to involve a carefully orchestrated sequence of conformational changes to ensure one-way and selective transport. But it has been found that sometimes water and ions get through too, and the question arises of whether this is passive, osmotically driven ‘leakage’ or stoichiometric co-transport of these species. Emad Tajkhorshid and colleages at Illinois at Urbana investigate this question using MD simulations, for several classes of membrane transporters (J. Li et al., PNAS 110, 7696; 2013 – paper here). They find that these generally support states in which there are channels that permit passive water flux – but that this does not interfere with the coordinated vectorial transport of the primary substrate. Thus the transporters are imperfect, but not problematically so: as the authors put it, “Given the soft mechanical properties of transporter proteins, it comes as no surprise to observe harmless imperfections in the overall gating motions, which manifest themselves in the formation of water-conducting states. It would, of course, be a concern if these channels were large enough to leak the substrate, and/or long-lived to allow very large amounts of smaller species to permeate across the membrane. Neither of these aspects appears to be the observed in our results, because the leaky states are only large enough for small species such as water. Furthermore, it appears that these states only transiently rise during the transport cycle, an attribute that might make them difficult to capture experimentally.”

Solvation of small molecules in water has often been described using point charges affixed to the solutes to represent polarization effects. David Cerutti at Rutgers and colleagues present a quantum chemical approach for fitting such partial charges to solutes like amino acids (D. S. Cerutti et al., J. Phys. Chem. B 117, 2328; 2013 – paper here). They say that their approach, which represents an evolution from the simplest point-charge models of previous decades, predicts substantially more polarization of amino acids than earlier efforts using AMBER force fields.

More on urea-induced protein denaturation, this time from Michela Candotti of the Institute of Research in Biomedicine in Barcelona and colleagues, who use MD, SAXS and NMR data to develop an atomistic picture of the unfolded states and the energetics of unfolding and refolding (M. Candotti et al., PNAS 110, 5933; 2013 – paper here). They conclude that urea’s denaturing influence is a combination of kinetic (disrupting stabilizing intramolecular contacts) and thermodynamic (stabilizing the extended conformation) effects. As I understand it, the results support models based on direct interactions of urea and protein, while also revealing that the urea-unfolded state is rather different to the denatured conformation that exists in pure water.

Hydration is evidently important to DNA conformation, influencing the A-B transition, interactions with DNA-binding proteins, and perhaps even affecting shape in a sequence-dependent manner. There is some evidence that the hydration water of DNA has collective dynamics of a glassy nature, and this notion is offered further support in inelastic neutron scattering experiments by Alessandro Paciaroni at the Università degli Studi of Perugia and colleagues (A. Paciaroni et al., J. Phys. Chem. B 117, 2026; 2013 – paper here). They find that at 100 K the large-wavevector scattering from water, related to coherent excitations, seems to imply a character related to amorphous ice. In other words, the interactions with DNA significantly alter the structure and dynamics of the interfacial water.

Melittin, one of the multi-subunit peptides that seems to aggregate by dewetting, is a component of been venom that acts as an antimicrobial. The melittin tetramer forms a pore that inserts into lipid membranes, and Max Berkowitz and colleagues at UNC now suggest that its effect is to create transient water-permeable channels, making the membranes leaky (K. P. Santo et al., J. Phys. Chem. B 117, 5031; 2013 – paper here). The technique they use for MD simulations can handle timescales of up to microseconds, and they show that the melittin peptides gradually aggregate into a kind of wedge that punctures the membranes and allows water to pass through, before falling apart again.

[FeFe] hydrogenase catalyses hydrogen-ion reduction to gaseous hydrogen, and could therefore be a useful biocatalyst. Simulations by Martin McCullagh and Greg Voth at Chicago now show that it seems to work by coupling electron transfer to proton transfer along a previously unknown water channel accessing the active site (J. Phys. Chem. B 117, 4062; 2013 – paper here).

There are seemingly strange rumours that fully miscible liquids are inhomogeneous on length scales of hundreds of nanometres, a conclusion suggested by some light-scattering and small-angle neutron-scattering studies. For example, Marián Sedlák of the Slovak Academy of Sciences reported such a claim in a series of papers in 2006 (e.g. J. Phys. Chem. B 110, 4329). Could these be solute clusters, or perhaps nanobubbles stabilized by adsorbed solute? Sedlák and Dmytro Rak now investigate that latter possibility (J. Phys. Chem. B 117, 2495; 2013 – paper here). They look at a range of solutes: magnesium sulphate, citric acid, urea, and t-butyl alcohol, and find no significant differences in the scattering from normal and degassed solutions, apparently ruling out the nanobubble interpretation.

Hydration and thermostability

What makes thermophilic proteins stable? And in particular, does hydration play a role? Those questions are examined by Fabio Sterpone of the Université Paris Diderot and colleagues (O. Rahaman et al., Phys. Chem. Chem. Phys. 15, 3570; 2013 – paper here). They study two homologous proteins, one from E. coli and the other from the thermophile Sulfolobus solfataricus, using MD simulations. The average water dynamics at the protein surface is the same in both cases, despite their different amino acid compositions, being slowed by a factor of 3-5 relative to the bulk. The authors conclude that this slowdown is primarily a geometric effect due to excluded volume, which explains why the protein sequence has little influence. This doesn’t exactly answer the initial question about thermostability – but it does suggest that there is nothing special about thermophilic proteins in terms of their hydration.

More on dewetting as a mechanism for protein folding and stability. Ruhong Zhou, Robert Matthews and their colleagues at Columbia have simulated a TIM barrel protein and found drying inside clusters of hydrophobic residues, signified by strong fluctuations in water density (P. Das et al., JACS 135, 1882; 2013 – paper here). In particular, a hydrophobic cluster (ILV) at the N-terminus shows drying that is weakened or suppressed by substituting some of the hydrophobic amino acids for less hydrophobic ones. A cluster at the C-terminus, meanwhile, seems to experience only partial drying that is unaffected by such substitutions. Experiments on the structure and stability of wild-type and mutant versions seem to back up these conclusions. The authors note that ILV clusters are common in several other protein motifs too.

Huib Bakker and colleagues at the FOM Institute in Amsterdam have used Förster resonant energy transfer between water OH stretches to find out where the water hydrating lipid membranes actually is (L. Piatkowski et al., J. Phys. Chem. B 117, 1367; 2013 – paper here). Somewhat surprisingly, they find that, even at rather low hydration levels, the water molecules are not evenly distributed among the lipid head groups, but form nanoscale clusters with an average intermolecular distance of 3.4 Å.

Our picture of the water surface continues to evolve. Is the water here structurally and dynamically different from that in the bulk? That idea is supported by experiments by James Skinner and colleagues at Wisconsin in simulations using a new three-body water potential (Y. Ni et al., PNAS 110, 1992; 2013 – paper here). They calculate that the hydrogen-bond switching dynamics are retarded at the water surface by a factor of about 3, although the rotational dynamics are actually a little faster. They say that vibrational 2D sum-frequency generation spectroscopy should be a good experimental method for investigating these dynamics, and calculate what the spectra should look like.

To make optimal use of plant cellulose as a feedstock for biofuels such as ethanol, it’s necessary to get it into aqueous solution. But cellulose is hard to solvate, which is why it is important to understand its hydration structure. Sylvia McLean at Oxford and colleagues have used neutron diffraction to look at the aqueous solvation of the disaccharide cellobiose, and find that there is (as has been suggested) a hydrogen bond across the glycosidic bond linking the two sugars (W. B. O’Dell et al., PLoS ONE 7, e45311; 2012 – paper here). There is competition from water molecules for the oxygen acceptor in this bond, however, with average occupancy of 50% for both water and the intramolecular OH donor.

More evidence that a hydrogen-bonded cluster of water molecules at the catalytic site plays a crucial role on photosynthesis: Bridgette Barry and colleagues at Georgia Tech use EPR to look at the rate of reaction of an intermediate neutral radical in the proton-coupled electron transfer that leads to oxygen evolution at the reaction centre of photosystem II, which contains a catalytic Mn4CaO5 cluster (J. M. Keough et al., J. Phys. Chem. B 117, 1296; 2013 – paper here). They find that this network is rearranged during the transition between the S0 and S2 states of the catalytic cycle, and that ammonia slows oxygen evolution because it disrupts the network by displacing water.

Suzi Jarvis and colleagues at University College Dublin have been using scanning probe microscopy for some time to probe hydration forces, and in their latest contribution they use a technique called frequency modulation AFM to look at hydration forces at the interface of mica and an electrolyte (J. I. Kilpatrick et al., JACS 135, 2628; 2013 – paper here). By ‘hydration force” here they mean the monotonically decaying force with a characteristic length of a few Å, without regard to its precise origin. They find that, relative to pure water, ions introduce or accentuate oscillations in the force as a function of distance due to the formation of distinct hydration layers. They point out that there are implications for obtaining atomic-resolution AFM images in aqueous saline solution.

Uzi Landman, Gary Schuster and colleagues at Georgia Tech offer a fascinating insight into the role of hydration water around DNA in the oxidation and consequent mutation of A/T-rich regions, which my be significant in the early stages of carcinogenesis due to stalling of replication (R. N. Barnett et al., JACS 135, 3904; 2013 – paper here). Their experiments and simulations indicate that oxidation of adenine leads to proton-coupled electron transfer to thymine, mediated by a water wire. This process can explain why nearly all the mutation due to such an oxidation event happens at thymine.

It’s sobering to realise that even now the reasons for the anomalous behaviour of water’s thermodynamic response functions, such as the divergence in the heat capacity and compressibility at low temperature, are still unknown. Francesco Mallamarce, Carmelo Corsaro and Gene Stanley discuss this issue with reference to experimental data on the power spectrum of sound velocity (F. Mallamarce et al., PNAS 110, 4899; 2013 – paper here). They conclude that the anomalies are associated with a structural transformation due to the appearance of an extended hydrogen-bonding network, which gives rise to viscoelastic behaviour in the liquid.

Also on bulk water, what happens to water’s structure above its critical point has been studied using X-ray Raman spectroscopy by Christoph Sahle of the Technical University of Dortmund and colleagues (C. Sahle et al., PNAS 110, 6301; 2013 – paper here). They find that, as one might expect, distortions of the hydrogen bonds are significant above the critical point, and the average coordination of each molecule decreases to just 0.6 at 600 oC and 134 MPa.

How antifreeze proteins work

A relatively short catch-up, with much more to come…

Martina Havenith at Bochum and her colleagues have extended their previous work on the mechanism of antifreeze proteins. Previously they looked at an antifreeze glycoprotein and found, using terahertz spectroscopy, that ice-binding by the protein seems to involve a long-range retardation of water H-bond dynamics, extending up to 2 nm from the molecular surface (S. Ebbinghaus et al., JACS 132, 12210; 2010). Now they find a similar effect operating for the antifreeze protein (AFP) of the fire-coloured beetle D. canadensis, a hyperactive insect (K. Meister et al., PNAS 110, 1617; 2013 – paper here). This contrasts, however, with the mechanism of another class of AFP, called wfAFP-1, which seems to operate only by short-ranged water binding to surface OH groups (S. Ebbinghaus et al., Biophys. J. 103, L20; 2012). As the authors say, “Nature is probably more inventive than initially thought and makes use of short- and long-range water perturbation to varying degrees in different classes of AFPs”.

Meanwhile, Ido Braslavsky of Ohio University and colleagues have used microfluidic methods to study the effects of AFPs on ice nucleation, and find that the growth of ice crystals is inhibited by the irreversible surface binding of the proteins (Y. Celik et al., PNAS 110, 1309; 2013 – paper here). These results help to rule out suggestions that direct binding of the AFPs to ice is not necessary to their mode of action.

More cases of water assisting receptor-substrate recognition and catalytic activity. First, Stephen Neidle at UCL and colleagues find that a cluster of 11 water molecules in an AT region of the minor groove of DNA seems to support the binding of three different small-molecule ligands (D.-G. Wei et al., JACS 135, 1369; 2013 – paper here). This cluster stabilizes the ligand by hydrogen bonding, and is also linked to (but distinct from) the well-known spine of hydration in B-DNA. Slight differences in binding mode with this cluster seem to account for the differences in binding affinity of the ligands: in other words, it is the water network that is calling the shots.

Second, Xiaoqing Wang and Hajime Hirao at Nanyang Technological University in Singapore say that the catalysis of myo-inositol monophosphatase (IMPase, a potential target for lithium treatment of bipolar disorder) is dependent on two bound water molecules (J. Phys. Chem. B 117, 833; 2012 – paper here). One provides the hydroxide ion that attacks the bound substrate. The other, coordinated to a magnesium ion, facilitates proton transfer leading to the product.

There seems to be an increasing perception that understanding the collective vibrations of proteins – their softness or rigidity – could offer insights into their enzymatic activity. Sow-Hsin Chen at MIT and coworkers support that view with an X-ray scattering study of the collective modes of hydrated lysozyme (Z. Wang et al., J. Phys. Chem. B 117, 1186; 2013 – paper here). They find that at low hydration levels both the collective ‘soft’ phonon modes and the enzymatic activity are much weaker or absent, suggesting a causal relationship: an indicator of the now familiar plasticizing effect of hydration.

C. Cametti at “La Sapienza” University of Rome and colleagues consider another aspect of protein hydration: how high concentrations of the protein (again lysozyme) can lead to clustering and a consequent decrease in average hydration number (C. Cametti et al., J. Phys. Chem. B 117, 104; 2013 – paper here). Their measurements of the dielectric spectra from 500 MHz to 50 GHz, which probe orientational relaxation, are consistent with this hypothesis of clustering into small aggregates at concentrations above about 100 mg/mL, which was first proposed by Stradner et al. (Nature 432, 492; 2004).

There is now some debate about whether the proposed two metastable liquid phases and associated critical point claimed on the basis of some simulations is real or not. That has been disputed by David Limmer and David Chandler at Berkeley, who extend their previous negative results using several different water potentials in a new preprint. I have written a commentary on the issue here.