Hot spots and cavities

Ariel Fernández is developing a picture of biomolecular hydration that might be considered to make it analogous to the standard picture of liquid-solid interfaces in terms of surface free energy. That’s to say, he defines a measure called the epistructural interfacial tension (EIT) which plays the role of a kind of surface tension for proteins in water, being increased where hydration is imperfect (that is, where hydration water molecules have a reduced number of hydrogen-bond contacts) relative to the bulk. This EIT can then be considered to promote protein-protein associations where this can lower the total free energy, via hot spots on the protein surface where the EIT is elevated: regions of high surface free energy, you might say. Ariel has developed this notion previously (Proc. R. Soc. A 467, 559; 2011), and in a new paper (Phys. Rev. Lett. 108, 188102; 2012 – paper here) he explains how to derive the ‘water coordination field’ around a solute particle that minimizes the EIT. This reduces to the macroscopic surface tension in the large-curvature limit. Hot spots for protein association correspond to those positions on the surface that reduce the EIT most significantly on binding.

I have seen few more striking examples of the involvement of ‘structured’ hydration water in enzyme action than that supplied by Ursula Rothlisberger at EPFL in Lausanne and colleagues (E. Brunk et al., JACS 134, 8608; 2012 – paper here). They use classical and quantum MD to look at the mechanism of the DNA-repair enzyme MutY, which excises a DNA base damaged by hydroxyl radical oxidation. The first (rate-determining) step of the process involves protonation of the N7 of adenine, which is assisted by a highly organized catalytic cluster of five water molecules, as shown here. The authors suggest that the particular arrangement of hydrophobic and hydrophilic residues around the water cluster act as a ‘water trap’ to ensure that these waters have long residence times.


How hydration influences and perhaps dictates pressure- and cold-induced protein denaturation has been much studied, but is still in debate. Payel Das of IBM Yorktown Heights and Silvina Matysiak of the University of Maryland shed some light on this question with MD simulations of the denaturation of a model 32-mer hydrophobic polymer (J. Phys. Chem. B jp211832c – paper here). They find that folding to the compact state happens via a hairpin-like configuration with a vapour bubble at the elbow, and that high pressure suppresses this process by reducing water fluctuations – observations that seem rather consistent with the current view of dewetting-induced hydrophobic collapse. Cold, meanwhile, leads to enhanced tetrahedral ordering of the hydration shell, which also suppresses the collapse.

Ah, but here’s the thing: cavities – or if you like, vapour bubbles – inside a protein have been generally found to be destabilizing, tending to induce denaturation, as discussed in a Commentary article in PNAS by Brian Matthews of the University of Oregon (PNAS 10.1073/pnas.1204795109 – paper here). This piece is motivated by a paper by Roche et al. (PNAS 109, 6945; 2012 – paper here) looking at the influence of cavities on pressure denaturation, specifically on the pressure-induced unfolding of SNase mutants that have appreciable dry cavities in their interiors. They find that the cavities play a dominant role in the unfolding process via the reduction in protein volume on denaturation.

The coupling of protein and hydration-shell dynamics is the subject of a paper by Jeremy Smith and colleagues at Oak Ridge (L. Hong et al., Phys. Rev. Lett. 108, 238102; 2012 – paper here). They follow the dynamics up to ns timescales in MD simulations and neutron-scattering measurements of lysozyme, and find that the protein dynamics can be decomposed into three components: diffusion within a local potential well, methyl rotations and nonmethyl ‘jumps’ – which I take to be jumps of localized diffusional chain motion from one potential well to another. Hydration seems to increase the probability of these latter jumps, but it also broadens the volume accessible for localized diffusion – in effect making the protein ‘softer’. Moreover, this effect propagates from the solvent-accessible surface deep into the ‘dry’ protein interior, loosening up the entire molecule.

Coupling of hydration dynamics is also the topic of a study by Kevin Kubarych and colleagues at the University of Michigan (J. T. King et al., J. Phys. Chem. B jp300835k – paper here). They use ultrafast IR spectroscopy to probe water dynamics near the surface of two variants of lysozyme. As previous studies have indicated, these dynamics are highly heterogeneous in space, and can vary from bulk-like to highly retarded, the latter being found at extended hydrophobic surfaces and being due to reduced hydrogen-bonding exchange. Using solvent-exchange, they also find that the liberation of this water drives the association of small molecules (and by extension, perhaps other proteins) at the hydrophobic protein surfaces.

Bridging water molecules often play an important catalytic role in receptor-substrate interactions. But not, it seems, in the mechanism of threonyl-tRNA synthetase: James Gauld of the University of Windsor in Canada and colleagues show that, contrary to previous suggestions, a water molecule here seem to serve no useful role, but only disrupts the process (E. A. C. Bushnell et al., J. Phys. Chem. B 116, 5205; 2012 – paper here).

The effect of progressive hydration of a protein from an essentially dry state is studied by Oleg Boyarkin and colleagues at EPFL in Lausanne (N. S. Nagornova et al., Science 336, 320; 2012 – paper here). They form gas-phase clusters of the macrocyclic peptide gramicidin S complexed with various numbers of water molecules, from 1 to 50, and characterize the interactions using vibrational spectroscopy to monitor hydrogen-bonding. The effect of progressive hydration is most pronounced for the first two water molecules, which disrupt intramolecular non-covalent (amine cation to pi) bonds and cause significant elongation of the peptide, bringing it into a conformation close to that of the fully hydrated state.

An interesting preprint on dewetting-induced hydrophobic attraction by Naiyin Yu and Michael Hagan at Brandeis (arxiv:1206.1828 – paper here). The latest view of this picture (see Patel et al., J. Phys. Chem. B 116, 2498 (2012) – Hagan is a coauthor of this paper) posits that proteins that self-assemble via hydrophobic interactions tend to sit close to a dewetting transition, which – if it doesn’t happen spontaneously – might therefore be induced by small perturbations. Yu and Hagan support this idea with simulations of the dimerization of the HIV capsid protein. While the wild-type subunits assemble only by gradual expulsion of water, mutations enacted in silico to replace three hydrophilic residues at the interface with hydrophobic ones can trigger dewetting – and moreover that they do so in a way that reveals the cooperativity of drying and the sensitivity to surface topography. As the authors argue, “Since the CA-C dimerization interface is typical of protein binding surfaces, our results raise the possibility that many proteins sit near a dewetting transition”.

A simple model of the hydration of a hydrophobic particle is presented by Ken Dill of Stony Brook and colleagues in Ljubljana (M. Luksic et al., J. Phys. Chem. B 116, 6177; 2012 – paper here). The model is two-dimensional, with water molecules modelled as ‘Mercedes Benz’ circular particles that engage in trigonal hydrogen bonding – simple enough to be solved almost analytically. The aim here is to probe the effects of hydrogen bonding and van der Waals interactions on the thermodynamics of hydration. The model is able to qualitatively capture the trends seen experimentally, such as the temperature dependence of thermodyamic functions such as the heat capacity and the effect of changing solute size, establishing it as a computationally cheap starting point for more sophisticated 3D models.

Solvation of a simple model hydrophobe is also the subject of a study by Pier Luigi Silvestrelli and colleagues at the University of Padova (L. Rossato et al., J. Phys. Chem. B 116, 4552; 2012 – paper here). They perform first-principles calculations for the hydration of methane, and find that the hydrophobic solute does not (as has been sometimes suggested) lead to a substantial slowing of the dynamics of the few water molecules in the first hydration shell, but rather to a slight (less than twofold) slowing of many water molecules. This slowing is caused by retardation of hydrogen-bond exchange.

More problems for the notion of ions as structure-makers and breakers comes from a study by Nuno Galamba of the University of Lisbon, who uses MD simulations to look at the perturbations of water tetrahedrality in the coordination shells of sodium halides (J. Phys. Chem. B 116, 5242; 2012 – paper here). Galamba points out how confused the current kosmotrope/chaotrope picture already is, for example because the effects of an ion on ‘water structure’ can be quite different in different hydration shells. Galamba concludes that a classification of kosmotropes/chaotropes based on the first hydration shell does not necessarily relate to what happens beyond that, so that the position of an ion in the Hofmeister series does not necessarily relate in a simple or transparent way to its overall effect on the hydrogen-bonded network of water. In terms of the implications for biomolecules, the paper’s final sentence provides, I think, a fair statement of the emerging consensus: “ion−protein interactions are likely to be much more significant than ion-induced long-range water structure perturbations, in agreement with the current view about Hofmeister ion effects on protein stability.”

Not quite the normal “water in biology” fare, but certainly warranting that general description, is a paper by Philippe Marmottant at the University of Grenoble and colleagues on water under tension (O. Vincent et al., Phys. Rev. Lett. 108, 184502; 2012 – paper here). Such water occurs in the xylem of trees, where it is drawn up by transpiration from leaves. I recently saw a very nice presentation on this subject by Adrian Strook at Cornell, who is making studies along similar lines to that reported in this paper. To model the plant system, Marmottant and colleagues use a hydrogel perforated with a regular array of cavities about 50 microns across. The main question they set out to investigate is what drives cavitation when the water pressure becomes increasingly negative – in plants, cavitation threatens to disrupt water transport. The tension here is generated simply by letting water evaporate from the gel. It seems that, when a bubble is nucleated (either spontaneously or stimulated by laser), it grows quickly to a quasi-equilibrium radius and then grows rather slowly larger – a very different process from cavitation in a bulk liquid, where cavitation is typically followed by rapid, energetic and potentially destructive bubble collapse (for example leading to sonoluminescence).

Now with pictures (what took me so long?)

Despite that previous post, I have found a bit of time while on travel, and so here's a catch-up.

Now here’s something very interesting. The ‘dewetting’ theory of protein assembly, according to which hydrophobic surfaces are drawn together at small separations by a cooperative, abrupt drying transition, has been much debated. Simulations have shown that dewetting can happen for flat hydrophobic plates, and also for hydrophobic polymers, but it seems to be rather rare for real proteins, in part because of their chemical heterogeneity: only a few hydrophilic groups on the surfaces are sufficient to suppress the transition, leading instead to gradual molecule-by-molecule expulsion of water. Now Song-Ho Chong and Sihyun Ham of Sookmyung Women’s University in South Korea have looked carefully at one particular protein assembly process: the dimerization of amyloid-beta, a 42-residue peptide linked to Alzheimer’s disease (PNAS doi:10.1073/pnas.1120646109 – paper here). The researchers study the aggregation with MD, and then apply the integral-equation theory of liquids to the simulated conformations to deduce the thermodynamics of solvation. They see no dewetting, but instead two distinct regimes of assembly. At relatively large separations it is the hydrophilic groups that seem to drive the attraction of the monomers, via an enthalpic interaction. Then as the protein surfaces come into contact, there is a switch to an entropically driven process that is water-mediated and involves dehydration of both hydrophobic and hydrophilic groups. I have not before seen anything much like this picture of protein assembly, although it does somewhat bring to mind the water-mediated contacts over long distances (1 nm or so) adduced in protein folding by Papoian et al. (PNAS 101, 3352; 2004).

The dewetting or capillary-evaporation picture of hydrophobic assembly has so far paid scant attention to the rate of water explusion in the hydrophobically confined region. Sumit Sharma and Pablo Debenedetti fill this gap, with some striking conclusions (PNAS 109, 4365; 2012 – paper here). They find that the (predominantly enthalpic) free-energy barrier to evaporation depends sensitively on the separation between the surfaces (assuming here a slit-like geometry), such that a change in separation from 9 to 14 Å results in a rate of evaporation that differs by ten orders of magnitude.

Another view of protein aggregation is offered by Alfonso De Simone at Imperial College in London and colleagues, who have looked at the formation of amyloid structures with non-pathological, functional significance: the hydrophobins that form robust protein coats on fungal spores, making them resistant to wetting (A. De Simone et al., PNAS doi:10.1073/pnas.1118048109 – paper here). What is striking about these proteins is that they are ‘designed’ to remain soluble unless they come into contact with a hydrophobic-hydrophilic interface, such as the air-water interface. The simulations in this study suggest that a section of the peptide chains that is highly flexible and disordered in bulk solution acts to suppress aggregation in that environment. At the air-water interface the proteins have drastically reduced access to his ensemble of conformations.

In contrast to this view, Martin Scholtz of Texas A&M and colleagues say that keeping proteins soluble seems to involve negative surface charge (R. M. Kramer et al., Biophys. J. 102, 1907; 2012 – paper here). They reach this conclusion by studying the effects on solubility of a range of proteins of adding two types of precipitant (that do not denature the proteins they precipitate): ammonium sulphate and polyethylene glycol 8000. They think that the observed correlation between solubility and surface negative charge is probably due to the strong water-binding propensity of glutamate and aspartate groups.

It is in fact the strong hydration promoted by these very groups that has been proposed as the reason for the stability of proteins in halophilic organisms, which have to resist the unfolding and aggregation that high salt concentrations might ordinarily induce. Bertil Halle and colleagues at Lund have probed this idea by looking at the water dynamics hydrating a halophilic protein Kx6E using oxygen-17 NMR (J. Qvist et al., J. Phys. Chem. B 116, 3436; 2012 – paper here). They find that these dynamics are not significantly different from those of the non-halophilic counterpart of this protein, challenging the notion of very tightly bound water molecules in the hydration sphere of halophilic proteins and, for that matter, the suggestion that cell water in extreme halophiles has slower dynamics than that in other cells (M. Tehei et al., PNAS 104, 766; 2007).

In a theoretical study of the hydration of hydrophobic surfaces using density-functional theory, Y. Djikaev and E Ruckenstein at SUNY at Buffalo support this notion that the hydration free energy here is primarily enthalpic (J. Phys. Chem. B 116, 2820; 2012 – paper here). They say that hydration is generally unfavourable at room temperature, and that the hydration free energy increases with increasing temperature, partly because entropic effects become increasingly negligible.

What is the hydrophobic interaction anyway? According to Snyder et al. (PNAS 108, 17889; 2011), we should start thinking in terms of a multiplicity of such effects; the work above seems to offer some endorsement for that view. Classically, the signature of a hydrophobic effect in protein folding has been considered to be a large difference between the heat capacities of the native and unfolded forms. But Robert Baldwin at Stanford, reviewing the history of how hydrophobic effects have been identified and rationalized (PNAS 10.1073/pnas.1203720109; 2012 – paper here), suggests that new definitions of the hydrophobic free energy are needed.

Some months ago, Umeda et al. reported a very beautiful crystal structure of photosystem II at 1.9 Å resolution, including a detailed picture of the hydration sphere (Nature 473, 55; 2011). They argued that several features of the hydration water distribution suggest that it forms a hydrogen-bonded network with specific roles in the catalytic mechanism of water splitting, such as relaying protons or other water molecules to active regions. Brandon Polander and Bridgette Barry at Georgia Tech have now looked at this idea in some detail (PNAS 109, 6112; 2012 – paper here). They focus on the oxygen-evolving complex, containing a Mn-Ca complex in which two water molecules ligate Mn and two Ca. By perturbing this H-bonded network with ammonia (which may substitute for water) and trehalose (which reverses the effect of ammonia), the authors deduce that the network is indeed critical for the photo-oxidation of water leading to oxygen evolution.

More on urea-induced protein denaturation. Yuguang Mu of Nanyang Technological University in Singapore and colleagues use MD simulations to study the salting in/out effects of urea and NaCl on amino acids and on a small amide mimicking a peptide backbone (W. Li et al., J. Phys. Chem. B 116, 1446; 2012 – paper here). They find, in line with experiments, that NaCl induces salting out, and urea salting in. By calculating the thermodynamic driving forces of these effects, they deduce that urea’s effect is caused not by the formation of hydrogen bonds with the peptide side-chain (or here, their mimics) but by an attraction mediated by van der Waals forces. This supports the idea that urea denaturation is mediated by direct interactions with the protein, but challenges the view that these interactions are hydrogen-bonding ones.

Susmita Roy and Biman Bagchi of the Indian Institute of Science in Bangalore present a picture of protein hydration in terms of water molecules in the hydration shell that are separated from the bulk by a free energy barrier against their escape (J. Phys. Chem. B 116, 2958; 2012 – paper here). They present MD simulations of hydration of the chicken villin head piece, which show that the residence time of hydration waters is strongly sensitive to secondary structure. In particular, a small subset of waters in one particular location, constituting about 5-10% of the total hydration shell, have residence times of around 100 ps. They seem to form a cluster that plays a central role in stabilizing the protein conformation by clamping together two of the protein’s three helices. Here then is another example of ‘quasi-bound’ waters becoming what one might consider as an intrinsic (albeit ‘loose’ and exchangeable) part of a protein’s secondary structure.

The effect of molecular crowding on hydration, for too long neglected, seems now to be getting the attention it deserves. The latest study comes from Michael Feig, currently at RIKEN in Kobe, and colleagues (R. Harada et al., JACS 134, 4842; 2012 – paper here). They have looked at how crowding affects water structure in concentrated solutions of a segment of streptococcal protein G by itself and combined with chicken villin head piece. They find that differences in local water density close to the dilute and crowded (>30% protein by volume – a physiological relevant situation) proteins are more pronounced beyond the first hydration shell; in particular, relatively ordered regions in the dilute case are often disrupted by crowding. What’s more, water diffusion is significantly reduced in the crowded case, and the dielectric constant is reduced. That latter change is especially significant, since it should reduce hydrophobic attraction and, by reducing electrostatic shielding, enhance the strength of hydrogen bonds and salt bridges. This in turn might be expected to stabilize secondary structure while destabilizing tertiary structure. These issues must surely be taken seriously in cell biology: how have proteins adapted, as one must assume they have, to withstand these likely perturbations in the strength of the forces that hold them together? What would this imply for protein-ligand binding constants and dissociation rates?

Proteins undergo slow conformational fluctuations, but they also exhibit higher-frequency collective vibrations akin to those seen in disordered matter. What role, if any, does the solvent play in these fast vibrations? Alessandro Paciarone at the Università degli Studi of Perugia and colleagues have investigated this issue by using terahertz spectroscopy to probe the rapid collective vibrations of essentially dry maltose binding protein (A. Paciarone et al., J. Phys. Chem. B 116, 3861; 2012 – paper here). In the dry state these motions tend to be faster, suggesting that as, one might expect, a reduction in hydration brings about an increase in rigidity.

The distribution of many ions in aqueous solution is perturbed at the air-water interface, where the ions may be either selectively adsorbed or depleted. It is not yet clear what drives this segregation, but a paper by Richard Saykally and colleagues at Berkeley sets out to elucidate that (D. E. Otten et al., PNAS 109, 701; 2012 – paper here). They use resonant UV SHG spectroscopy to look at the distribution of thiocyanate ions (classified in the old-style Hofmeister schemes as a typical ‘chaotrope’) at the interface as a function of temperature, in order to measure the thermodynamic variables. They find that the adsorption enthalpy and entropy changes are both negative. The former reflects a balance between hydration and surface energies of the solvent (for example, how much an ion ruptures solvent-solvent hydrogen bonds), whereas the entropy change seems to result from the way adsorbed ions alter capillary-wave fluctuations at the surface. An ion’s Hofmeister effect should therefore reflect a balance of these two, essentially independent factors.

The homogeneity or otherwise of aqueous solutions, particularly those of alcohols and polyols, is a subject of ongoing debate. There seems good reason to suspect that even rather simple alcohols show a significant degree of aggregation. This seems to be true of 2-butoxyethanol, as revealed in simulations by Rini Gupta and G. N. Patey of UBC in Canada (J. Phys. Chem. B 115, 15323; 2011 – paper here). They find that aggregates of the alcohol begin to appear at mole fractions (X) of more than about 0.005, and by X~0.04-0.02 these are micelle-like and rather large, so that large simulation systems (around 32,000 molecules) are needed to accommodate them. The size of the aggregates at these mole fractions – around 4 nm – is big enough to be seen in light-scattering and SANS experiments, as has already been reported.

There’s increasing interest on the structure, dynamics and thermodynamics of biological macromolecules at the air-water interface. Ozge Engin and Mehmet Sayar at Koç University in Turkey have looked at this issue for amphiphilic peptides designed to sit at such interfaces and to self-assemble into ordered systems and nanostructures (J. Phys. Chem. B 116, 2198; 2012 – paper here). Specifically, they look at small peptides designed to fold into beta-hairpins in solution, which could act as models for studying, e.g. amyloid formation. Perhaps unsurprisingly, the segregation of hydrophobic and hydrophilic residues at the water surface promotes hairpin formation above what is observed in bulk solution, and the molecules form well-ordered monolayers with anti-parallel stacking.

There’s a potential synergy here with a paper by David Vaux at Oxford and colleagues on amyloid formation at the air-water interface (L. Jean et al., Biophys. J. 102, 1154; paper here). They find that the interface promotes amyloidogenesis, largely due to a simple concentration effect: the (representative) amyloid peptides they study are surface-active, and so there is enrichment at the air-water boundary (i.e. this does not speak to the issue of whether, say, hydrophobic interactions are altered at the interface). The implications are partly cautionary: such surface effects could be complicating in vitro studies of amyloid formation and drug screening. But as the authors point out, complex interfaces are also present in vivo.

Forgive me if I’ve mentioned this before, but I think somehow I haven’t: there is an excellent summary of David Chandler’s ideas on dewetting and water fluctuations in a preprint with Patrick Varilly prepared for the International School of Physics "Enrico Fermi", Course CLXXVI - "Complex materials in physics and biology" in Varenna, Italy (a very lovely place), which happened back in July 2010. It’s at arxiv/1101.2235 (here), but doesn’t seem to have been published in a proceedings yet.

Small and often

My new resolution is to try to post more often and in smaller chunks, not least to avoid an impression of this site being moribund. With that in mind, I acknowledge that there is a fair bit of old stuff to catch up on which is not yet covered here. I also hope, when I have a moment, to find a way of posting that will make this blog accessible in China, which it is not at present because it seems the whole hosting network is blocked due to Google-related wrangles.

Water in protein hydration shells has retarded dynamics, for example in terms of reorientation. But how much and why? Reports vary, from slowing by a factor of a few to several orders of magnitude. Damien Laage and colleagues at ENS in Paris have tried to clarify the situation with simulations of lysozyme (F. Sterpone et al., JACS 134, 4116; 2012 – paper here). They find that most (80%) of the hydration water is slowed by a factor of just 2-3, and that the dynamics seem to be dominated by the same kind of activated jumps between H-bond acceptors as in the bulk. This slowdown seems to be due to an excluded-volume effect from the proximity of the protein surface, which reduces the number of transition-state configurations for reorienting jumps. The remaining water may be slowed to a greater degree, apparently due to water bound within clefts and pockets on the protein surface, where generally it is bound to H-bond acceptors.

Ben Corry and Michael Thomas at the University of Western Australia say that water plays a role in the selectivity of voltage-gated sodium channels (JACS 134, 1840; 2012 – paper here). Simulations based on a recent crystal structure show that unlike sodium, potassium ions can’t fit between a plane of glutamate residues with water molecules bridging the ion and the carboxylate groups – so these latter ions are excluded even though in principle they could pass through the pore with their complete hydration shell. This suggests that there are more subtle structural factors at work than (as has been suggested previously) simply the free-energy penalty of ion dehydration.

The hydrophobic effect and its role in the assembly of hydrophobic particles has generally been considered from the perspective of an equilibrium process, with no account of hydrodynamic factors. Bruce Berne and his coworkers at Columbia now seek to rectify this (J. A. Morrone et al., J. Phys. Chem. B 116, 378; 2012 – paper here). They simulate the interactions of two fullerenes in water, taking into account how molecular-scale hydrodynamics affects solvent density fluctuations and drying transitions. Perhaps unsurprisingly, a continuum picture breaks down at the smallest length scales : for example, the friction coefficient deviates from the continuum prediction at small particle separations, and can become non-monotonic due to layering. In general, these hydrodynamic effects can significantly reduce the diffusion-controlled rate constant for hydrophobic assembly. As the authors say, how such effects would become manifest in the crowded environment of the cell is another matter.

When is a protein unfolded? It’s not such an easy question as one might suppose: Sunilkumar Puthenpurackal Narayanan and colleagues in Japan say (Biophys. J. 102, L08 – paper here) that Catherine Royer and colleagues have recently found that staphylococcal nuclease can show a proton NMR signal at high pressures indicative of complete folding while Trp fluorescence data suggest significant unfolding. Narayanan et al. see something comparable for a subdomain of the transcription factor c-Myb R2 at high pressure and low temperature. This, they say, seems to be explicable on the basis that the protein remains folded but extensively hydrated, owing to water filling of a large internal cavity. This adds to the ongoing debate about the mechanism of pressure-induced denaturation, which some say is caused by the intrusion of water. More on this in a later post.

I wish I had a better grasp of a paper by Vladimir Sirotkin and Aigul Khadiullina of the Kazan Federal University in Russia on excess partial enthalpies of water and proteins (J. Chem. Phys. B 115, 15110; 2011 – paper here). But the key point seems to be that the progression from more or less dehydrated to fully hydrated states of several different proteins follow the same trajectory, at least in thermodynamic terms. The excess partial enthalpy is initially dominated by water, but then up to a weight fraction of 0.06 both protein and water contribute significantly. At this point the charged groups on the protein are hydrated and the proteins become flexible. And by a weight fraction of 0.5, hydration is complete and further changes are due only to contributions from the proteins. This seems loosely to be in accord with notions of a roughly universal ‘critical coverage’ for a protein hydration shell.

More dynamical transitions?

Francesco Mallamace at MIT/Messina and colleagues have used proton NMR to follow the rearrangements of water during the thermal (heat and cold) denaturation of lysozyme (F. Mallamace et al., J. Phys. Chem. B 115, 14280; 2011 – paper here). Since the chemical shift is considered to probe hydrogen-bonding interactions in the water network, tracking it as the temperature changes take the protein from the folded to the denatured states reveal some aspects of how changes in protein structure are mirrored by those of the hydration water. The researchers conclude that water plays an active role in the process, and that as denaturation proceeds, the average number of H-bonds with which each water molecule is involved changes correspondingly.

How glycerol acts as a cryoprotectant is the subject of a study by J. Towey and L. Dougan at Leeds (J. Phys. Chem. B jp2093862 - paper here). They use neutron scattering to investigate whether, as one hypothesis has it, dissolved glycerol perturbs water structure to modify the hydrogen bonding ability and suppress ice formation. The solution turns out to be well mixed, with many glycerol monomers, but there is no discernible perturbation of the first coordination shell of water. The second shell, however, is perturbed in a manner similar to that caused by elevated pressure. Quite what this means for “the ability of water molecules to form ice” isn’t clear (indeed, I’m not even too sure what that expression means – displacement of the phase boundary?), but Towey and Dougan conclude that any explanation will need to focus not simply on local perturbations (or changes to water’s ability to form hydrogen bonds) but on changes to the extended hydrogen-bonded network.

More on the mechanisms of osmolytes: Dave Thirumalai and colleagues at Maryland have studied the stabilization of compact peptide conformations by trimethylamine N-oxide (TMAO) (S. S. Cho et al., J. Phys. Chem. B 115, 13401; 2011 – paper here). They attribute it to direct interactions between TMAO and the protein surface, which exclude solvent there. This is then a kind of excluded-volume effect analogous to the stabilization of proteins by molecular crowding (in which there is entropic destabilization of the unfolded state) – making TMAO a ‘nanocrowding’ particle.

In a related vein, M. Paulaitis at Ohio State and colleagues describe an integral-equation approach to deduce preferential interactions between cosolvents, which they say can be used to deduce preferential interactions of cosolvents such as osmolytes, naturants and cryoprotectants locally on the surface of proteins (M. H. Priya et al., J. Chem. Phys. B 115, 13633; 2011 – paper here).

Personally, I find it hard to think about hydration in crowded environments, in which marcomolecules might disturb one another’s hydration shells and sometimes temporarily associate with one another in non-specific ways. Sergio Hassan and Peter Steinbach at NIH have tried to provide a context from framing this question (J. Phys. Chem. B 115, 14668; 2011 – paper here). One issue is how incomplete and anisotropic hydration might create electrostatic effects. Another is how solvation forces due to structured hydration shells (layering, for example) manifest themselves on hydrogen-bonding at solute-water interfaces. Using a continuum solvent model, they say that the electrostatic effects of solvent exclusion can have a strong impact on protein-protein binding. But I think it fair to say that at this point the paper is largely presenting the methodology for investigating the problem, rather than reaching general conclusions about how these aspects of crowding affect the molecular biology.

Confinement will, of course, do other things to water itself. Ivan Brovchenko and Alla Oleinikova have predicted that water in slit-like pores just 2.4 nm wide might undergo the liquid-liquid transition predicted in the metastable bulk at low temperature and high pressure (J. Chem. Phys. 126, 214701; 2007). Limei Xu and Valeria Molinero at Utah have now examined that idea in simulations using their mW model of water held within 1.5-nm diameter cylindrical pores (J. Phys. Chem. B 115, 14210; 2011 – paper here). This system is comparable to the pores of MCM-41 nanoporous silica, as used in recent experiments on confined water (e.g. L. Liu et al., Phys. Rev. Lett. 95, 117802 (2005)). They find no evidence for a first-order liquid-liquid transition, but note that smearing of discontinuous transitions is well known in pores (although not in fact inevitable), and therefore that this doesn’t rule out the existence of such a transition in the bulk. For their simulations of the bulk phase, they do see a possible signature of a L-L critical point – a locus of maximum compressibility – but can’t study this in detail because fast crystallization makes it impossible to equilibrate a metastable water phase in this region.

How do proteins remain dynamic while remaining soluble and resistant to aggregation? Fabrizio Chiti at the University of Florence, Chris Dobson at Cambridge, and their colleagues, have sought to answer this by combining NMR relaxation data, H/D exchange experiments and MD simulations (A. De Simone et al., PNAS 108, 21057; 2011 – paper here). They use the fruitfly acylphosphatase as their model system, and find that the wild-type protein has free-energy barriers that limit access to aggregation-prone conformations except under aggrtegation-prone conditions (addition of small amounts of trifluoroethanol). They sum up the situation nicely: “The sensitivity of the energy surfaces of proteins to minor perturbations supports the view that there is a delicate balance between functionality, stability, and solubility, which is encapsulated by the concept of ‘life on the edge’”.

Hydrogen bonds hydrating hydrophobic regions of a protein or peptide seem to have a greater strength than those in bulk water. It’s been suggested that this might be not so much because the H-bonds are genuinely strengthened but because the orientational preferences of H-bonds in such a situation result in a depletion of weaker, strained H-bonds, i.e. a change in the population (Zichi & Rossky, J. Chem. Phys. 83, 797 (1985)). Peter Rossky and colleagues have now explored this idea further using MD simulations of a 16-residue peptide (J. Phys. Chem. B 115, 14859; 2011 – paper here). They find support for the idea, namely, water is depleted of near neighbours around apolar groups and so samples lower-coordination configurations that are undistorted and unstrained. In other words, the phenomenon is primarly a kind of packing effect.

A new angle on Hofmeister effects is offered by Huib Bakker at FOM Institute for Atomic and Molecular Physics in Amsterdam, who have looked at the orientational dynamics of water around various ions (K. J. Tielrooij et al., J. Chem. Phys. B 115, 12638; 2011 – paper here). When the salt consists of a strongly hydrated ion and a weakly hydrated counterion, the water molecules hydrating the former have impeded orientational dynamics, making it strongly anisotropic. In that case, they say that hydration is ‘semi-rigid’ in the first hydration shell: affected along one vector but not along others. If both ions are strongly hydrated, such perturbations of water dynamics extend well beyond the first hydration shell.

And while we’re there: Daryl Eggers and colleagues at San José State University have taken a thermodynamic line of attack on Hofmeister by determining the molar water volumes in various concentrated electrolyte solutions (A. Y. Payumo et al., J. Phys. Chem. B 115, 14784; 2011 – paper here). They find that the solutions are highly nonideal, presumably because of strong competition under these conditions for hydration water. Moreover, the solubility of the small amide diketopiperazine follows the Hofmeister series for all the anions and cations studied, and the authors explain this on the basis that Hofmeister effects are governed by changes in the average free energy of the bulk aqueous phase – that is, if Hofmeister effects are a bulk phenomenon of water.

The tale of the low-temperature dynamical crossover for hydrated proteins and their hydration shells continues to get more complicated. Using dielectric spectroscopy and MC simulations, Gene Stanley, Giancarlo Franzese and their colleagues now report evidence of two such crossovers for the hydration water of lysozyme: one at about 252 K, the other around 181 K (M. G. Mazza et al., PNAS 108, 19873; 2011 – paper here). Marie-Claire Bellissent-Funel and her colleagues have previously seen something similar – two transitions at 220 and 150 K (J.-M. Zanotti et al., PCCP 10, 4865; 2008). Stanley et al. now ascribe the first of these to maximal fluctuations in the making and breaking of hydrogen bonds, and the second to maximal fluctuations in cooperative reordering of the H-bonded network.

Meanwhile, Sol Gruner and colleagues at Cornell report evidence for another protein dynamical transition right down at 110 K, which they say correlates with the transition of the hydration water from a high- to a low-density amorphous state (C. U. Kim et al., PNAS 108, 20897; 2011 – paper here).

Wilfred van Gunsterden at ETH and colleagues show how solvation free energies, as well as the free energies of protein-ligand binding and protein conformational dynamics, can be calculated using a new software package called GROMOS, which van Gunsterden and colleagues have introduced in a paper in press with Comput. Phys. Commun. (S. Riniker et al., J. Phys. Chem. B 115, 13570; 2011 – paper here).

Alan Soper has an intriguing paper in J. Phys. Chem. B (115, 14014; 2011 – paper here) in which he presents a new mixture model of water. This isn’t exactly a two-state model – the two forms are intimately mixed – but it postulates two populations of water molecules, each of which can form hydrogen bonds only with molecules of the other type and not with those of their own type. This is not, as far as I can see, intended as a literal representation of some molecular-scale distinction – both types of water molecule have identical structure – but is imposed as a device for introducing a three-body term into the interactions. The results show good agreement with structural studies using neutron and X-ray scattering, and give rise to a situation where water molecules are H-bonded to some of their neighbours but not others. This, Alan suggests, is perhaps why mixture models of water have been so enduring: not because there really are two distinct populations but because – if I’m understanding this correctly – the three-body terms have the effect of making it appear that way.

Michele Parrinello and colleagues at ETH have investigated the recombination of hydronium and hydroxide ions in water using ab initio MD simulations (A. Hassanali et al., PNAS 108, 20410; 2011 – paper here). They find that the mechanism is rather different from what has traditionally been assumed in terms of a Grotthuss mechanism. The researchers say that the Grotthuss mechanism serves to bring the hydronium and hydroxide to a distance of around 6 Å, when they are bridged by two water molecules as a ‘water wire’. But then there is a collective compression of this water wire that results in a concerted motion of three protons (rather than a series of distinct one-proton hops), converting both ions into water molecules.

Welcome to 2012

Lots to catch up on here, and while I’m not going to do that exhaustively now, here is an update to let you know that this blog will still be active in 2012.

I’ve recently written a News & Views article for Nature (478, 467; 2011 – here) about, among other things, the recent paper by George Whitesides’ group (PNAS 108, 17889; 2011 – paper here) on the hydrophobic effect in ligand binding – which, as discussed in the previous post, suggests that there is not any single ‘hydrophobic effect’ operating here, but a delicate and case-specific balance of enthalpic and entropic effects. They noted that in the example they studied, the ‘hydrophobic’ aspect of binding was driven largely by the enthalpic effect of displacing/rearranging water around nonpolar contacts. That notion is supported by a paper by Stephen Martin and colleages at the University of Texas at Austin, who find something rather similar for small peptide binding by the SH2 domain of the growth receptor binding protein Grb2 (J. M. Myslinski et al., JACS 133, 18518; 2011 – paper here). They say that increases in the nonpolar contact area don’t necessarily lead to entropic gains due to release of water, but have a primarily enthalpic influence on binding affinity.

How quickly drugs bind to their target molecules can have important pharmacological implications, but these kinetics are generally poorly understood, and thus to design rationally. Xavier Barril from the University of Barcelona and colleagues show using MD simulations that slow kinetics are often a consequence of the presence of buried polar atoms which form hydrogen bonds that are shielded from water, because of the slowness of dehydration/rehydration (P. Schmidtke et al., JACS ja207494u – paper here). In other words, the pace of events in binding can be set by the degree of water accessibility. They show that this effect can be predicted from structural data, and can thus potentially be accessible to design.

Knowing the hydration structure of proteins is important for small-/wide-angle X-ray scattering (SWAXS) studies of proteins not so much for its own sake but because the contribution of the solvent to the scattering must be subtracted in order to extract information about the protein secondary structure. It’s easy enough to extract the contribution of bulk water, but correcting for the scattering of the hydration shell is complicated. Tobin Sosnick and coworkers at the University of Chicago now show two ways to do this (J. J. Virtanen et al., Biophys. J. 101, 2061-2069; 2011 – paper here). One is to deduce the solvation structure by full MD simulations, which they say gives results that match the SWAXS data closely. But they have also developed a much more computationally less intense solvation model called HyPred, which gives a scattering profile that agrees well both with MD and with experiment.

Bernhardt Trout and his coworkers at MIT present an illustration of just how complex the interactons of proteins and ions can be (D. Shukla et al., JACS ja205215t – paper here). Complex ions such as guanidinium create a particularly intricate picture. Gdm+ can destabilize proteins via the formation of hydrogen bonds and electrostatic interactions, but when paired with an anion that is a hydrogen-bonding acceptor it can form clusters with the ions, which suppresses the effect. Molecules (such as arginine) with multiple Gdm+ groups are sometimes used to suppress protein aggregation, and can do so without compromising protein stability. Trout and colleagues investigate the effect of arginine oligomers (n=1-4) on protein aggregation and conformational stability for two different anions, chloride and sulphate. While monomeric arginine chloride is used as an aggregation suppressor, the n-mers only inhibit aggregation at low concentration – they actually accelerate it at moderate to high concentration. Meanwhile, the sulphates inhibit aggregation at all concentrations. And while the chlorides reduce protein stability, the sulphates enhance it. The researchers explain all this in terms of the balance between ion-ion and ion-protein interactions.

All this mirrors an increasing tendency to consider Hofmeister effects of ions on proteins in terms of direct interactions between the two species, rather than as indirect consequences of changes in hydration. Elena Algaer and Nico van der Vegt at the TU Darmstadt provide some support for this notion with a study of the salting-in and –out of small model amides by various sodium salts (J. Phys. Chem. B jp208583w – paper here). They say, for example, that the salting-in if NiPAM by NaI is mediated by interactions of iodide with the nonpolar groups. Such interactions also explain why, of all the salts studied, the iodide alone fails to induce hydrophobic collapse of polyNiPAM.

How does water permeate cell membranes? Its passage through the water-regulating membrane protein aquaporin is fairly well studied, but little is known about other water transporters. The bacterial sodium-galactose transporter vSGLT and its human homologue the sodium-glucose cotransporter hSGLT1 both have the potential to let water through. Jean-Yves Lapointe at the Université de Montréal and colleagues have used MC and MD simulations to show that indeed these protein pores can be filled with water (a pathway of about 100 molecules) which allows passive water permeation (L. J. Sasseville et al., Biophys. J. 101, 1887-1895; 2011 – paper here). This pathway depends on the proteins’ conformation: there is a constriction at one point which reduces the water bridge to a single-molecule chain which then ‘snaps’ at a ‘hydrophobic plug’, creating a 4.3 Å gap of low water density. But the resulting barrier to water permeation is conformation-dependent, and can be altered by varying the membrane potential. The passage of a sugar molecule also can bring water with it, but the mechanism of this is still open to debate.

Jhih-Wei Chu and colleagues at Berkeley conclude from MD simulations that the insolubility of cellulose in water is an entropic effect due primarily to the reduction of solvent entropy if the glucan chains in a fibril unravel (A. S. Gross et al., J. Phys. Chem. B 115, 13433; 2011 – paper here). There are some lessons here for how to solubilize cellulose in other solvents, such as ionic liquids.

Why and how do ions segregate at the air-water interface? This phenomenon seems well attested, with anions, especially large and polarisable ones, tending to accumulate at the interface. But the reason for this is still debated. Yi Qin Gao if the Beijing National Laboratory for Molecular Sciences and coworkers investigate the question with MD simulations, looking in particular at the differences in how anions and cations are solvated (L. Yang et al., J. Phys. Chem. B jp207652h – paper here). They argue that these differences are due to the charge distribution in the water molecules themselves, and that water can approach anions more closely than cations. They suggest that these differences in hydration account for why anions tend to populate the interface more readily.

And on the same topic, Pavel Jungwirth and colleagues have looked at how the hydration of guanidinium ions affects their orientation at the air-water interface (E. Wernersson et al., J. Phys. Chem. B jp207499s – paper here). Guanidinium is depleted at the interface, but the ions that do stay there are preferentially oriented parallel to the surface: in this configuration, it can sit at the surface without needing to break hydrogen bonds. Another way of looking at this is that the ions can take advantage of the deficit of hydrogen bonds between waters at the surface. The authors suggest that similar reasoning might account for the unexpected orientation of some arginine groups (with an analogous structure) in protein side chains.

Lum-Chandler-Weeks under the microscope

Shekhar Garde and his colleagues have shown in several recent publications how hydration of solutes and its consequences, such as conformational changes in polymers, can be significantly altered near the air-water interface. The more general effect of interfaces on hydrophobic phenomena is now probed by Garde along with David Chandler, Amish Patel and others (A. Patel et al., PNAS doi 10.1073/pnas.1110703108 – paper here). They look at how interactions between hydrophobic solutes of various sizes from sub-nm to several nm are altered close to the interface of water with self-assembled monolayers of various surface chemistries, from hydrophobic to hydrophilic. They find that the driving force for the assembly of hydrophobic particles is smaller near a hydrophobic surface than it is in bulk, and decreases with increasing temperature, in contrast to the bulk (and to hydrophilic surfaces). This implies that hydrophobic surfaces should act as catalysts for the unfolding of proteins. It might also account for how chaperonins work: their initially hydrophobic surfaces help misfolded proteins to unfold, and then ATP-driven conversion of the walls to hydrophilic release the unfolded protein from the wall so that it might fold again in bulk.

Misfolding in the context of Lum-Chandler-Weeks theory is also the subject of a paper by Ruhong Zhou and colleagues (Z. Yang et al., J. Phys. Chem. B 115, 11137 (2011) – paper here). They have looked at whether dewetting transtions play a role in the assembly of two amyloidogenic beta-sheets. For the peptides considered there are two conformations that lead to stacking of beta-sheet pairs: one containing water between the sheets in a 2D slab-like geometry, the other with the water in a 1D tube-like geometry. In both cases the sheets are brought together by a drying transition, but surprisingly this is stronger for the slab-like than the tube-like case. This is attributed here to the different surface roughness of the two packing modes: the ‘staggered’ packing in the slab-like case, which is rougher, disrupts the H-bonding network of the intervening water to a greater extent.

Some further new insights into the Lum-Chandler-Weeks dewetting mechanism for hydrophobic interactions are offered by two recent papers. Li and Walker appear to see the cross-over region between the mechanisms of hydrophobic hydration at small (<1 nm) and large (>1 nm) scales that this theory predicts, by measuring the free energy of hydration of individual monomers of various size in hydrophobic polymers, using an AFM to pull the chains out from a collapsed conformation (Li, I. T. S. & Walker, G. C. PNAS 108, 16527-16532; 2011 – paper here). At a monomer size of around 1 nm in the temperature region of 50 C or so, they find a switch in the hydration entropy from negative to positive (this crossover size reduces to just 3.5 Å at 150 C). Shekhar Garde and Amish Patel have published a commentary on it (10.1073/pnas.1113256108).

And Garde and Patel have joined forces with David Chandler and others in a preprint (arxiv.org/1109.4431 – paper here) that attempts to unravel the issue of why some protein subunits (such as melittin) seem to aggregate via dewetting while others (such as BphC) do not. They find that at model (SAM) hydrophobic surfaces, simulations show that the statistics of large-amplitude fluctuations in the density of interfacial water are altered relative to the Gaussian stats of both bulk water and water at a hydrophilic surface. In other words, despite similar average interfacial water densities, the fluctuations reveal the proximity of the hydrophobic interface to a dewetting transition. This tuning of the interfacial water is, they argue, common to biological systems, where it induces a strong sensitivity to small changes in conformation, allowing the system to take advantage of the phase transition in engineering biomolecular function (in a manner analogous to the finely balanced wetting or drying of ion channels for ‘vapour-lock’ gating). Although melittin and BphC lie on opposite sides of this transition, small modifications to both can tip the balance one way or the other. This offers what seems to me to be a persuasive argument that dewetting is relevant to hydrophobic aggregation even if it does not exactly provide the mechanism for it in all (or even in most) cases: the transition is, if you like, ‘there’ even if it doesn’t manifest itself.

Alenka Luzar and her colleagues have considered this issue from the perspective of whether or not cavitation can take place at the protein-protein interface (J. Wang et al., PCCP 10.1039/c1cp22082a – paper here). They present a lattice model which offers a fast method for predicting if cavitation can happen, and find that part of the surface of melittin is sufficiently hydrophobic to permit this on a timescale that is consistent with that seen in the earlier simulations.

Alenka and her colleagues have also examined how this putative crossover length-scale for hydration behaviour is influenced by charge on the solute (J. Stat. Phys. 10.1007/s10955-011-0337-1 – paper here). They conclude that, for moderate charge, the electrostatic contribution to the solvation free energy is in fact essentially independent of solute curvature, because of a compensation between counterion shielding and the dielectric screening of water – the solvation free energy remains more or less a function only of solute surface area.

To what extent protein-ligand binding requires an atomistic description of changes in hydration is a crucial question, not least for attempts to design synthetic ligands in drug development. Ulf Ryde at Lund University and colleagues have looked at this issue by comparing the ability of continuum methods to predict binding free energies for four different protein-ligand pairs with quite different degrees of solvent exposure at the binding site (S. Genheden et al., JACS ja202972m – paper here). They find that the continuum methods often perform badly, particularly for cases with a greater degree of solvent exposure. We need to know precisely where the waters are and where they go.

And that is somewhat elucidated by Nan-jie Deng at Rutgers University and colleagues for the case of two synthetic inhibitors of HIV-1 protease, Nelfinavir and Amprenavir (N.-j. Deng et al., J. Phys. Chem. B jp204047b – paper here). The binding of these drugs is apparently entropically driven, but the question is where that entropic contribution comes from. A classical view would be inclined to attribute it to the release of water from the binding cleft, but it seems that this isn’t so: these MD simulations suggest that any entropy gain there is more than offset by the restriction of ligand rotation and vibration on binding. Instead, the favourable entropic contribution seems to come from desolvation of the ligand.

A different view of water’s influence on bimolecular recognition is provided by Stacey Wetmore and colleagues at the University of Lethbridge in Alberta (F. M. V. Leavens et al., J. Phys. Chem. B jp205424z – paper here). They have looked at how water molecules can affect the pi-pi interactions between DNA and DNA-binding proteins. Solvating water molecules seem to have essentially no influence on the strength of pi-pi stacking for histidine and adenine, but do weaken the interaction if the histidine is protonated. The latter interaction, however, remains in all cases stronger than the former.

Protons seem to be delivered to membrane proteins such as the proton pump cytochrome c oxidase via some kind of surface-enhanced, two-dimensional transport at the membrane surface. This has been previously postulated as a series of jumps between ionisable groups (phosphate and carbonyl) at the membrane surface. But that notion is challenged by Peter Pohl at the Johannes Kepler University in Linz and colleagues, whose fluorescence measurements of proton transfer at membranes show that proton transport can be equally fast in the absence of ionisable groups (A. Springer et al., PNAS 108, 14461-14466; 2011 – paper here). They conclude that it is probably the network of interfacial water molecules that is responsible instead for the rapid proton motion: as they say, “water structuring at the interface seems to be mandatory for providing the pathway”.

It has of course been long thought that protons may be delivered to the interior of an enzyme via chains of water molecules. That process is studied by heme peroxidase by Emma Lloyd Raven and coworkers at the University of Leicester (I. Efimov et al., JACS ja2007017 – paper here). Kinetic isotope effects reveal that the proton pathway utilizes a Grotthus-like shuttling of protons along a pathway towards the ferryl oxygen that involves three bound waters and two arginine residues.

A model system for studying such proton transfer in confined geometry is reported by Bradley Habenicht and Stephen Paddison at the University of Tennessee in Knoxville (J. Phys. Chem. B jp205787f – paper here). They use MD simulations to look at how protons are transported within carbon nanotubes whose inner walls are functionalized with perfluoro sulphonic acid groups. If these groups are spaced far apart (~8 Å), they tend to be individually hydrated by clusters of water molecules with little interaction between them, and correspondingly reduced acidic proton dissociation. But there is also a pronounced effect of confinement at small nanotube diameters: in the smaller tubes there are stronger interactions between the walls and the water molecules, which can lead to break-up of the hydrogen-bonded network of waters linking the sulphonic acid groups. That network may be restored by polarized charges of fluorine atoms attached to the nanotube walls.

ATP hydrolysis in the active cleft of actin plays an important role in the state of its filamentous form F-actin, affecting its rigidity and its binding of regulatory proteins. There is water in this active site, but it hasn’t previously been clear what, if anything, it does. Marissa Saunders and Greg Voth at Chicago have clarified this through MS simulations based on the crystal structure (J. Mol. Biol. 413, 279-291; 2011 – paper here). They say that the ordered waters help the protein to flatten and brings about a conformational change that promotes ATP hydrolysis. These changes also stabilize the charge on the phosphate and accelerate the deprotonation of the catalytic water involved in hydrolysis. In short, the bound water helps to organize the active-site geometry.

Another nail in the coffin of ‘structure-making and –breaking’: Fabio Bruni and colleagues in Rome have looked at how local solvent structure around ions affects their influence on viscosity – specifically, on how changes in ionic concentration affect the viscosity of the solution (T. Corridoni et al., J. Phys. Chem. B jp202755u – paper here). Classically, the viscosity is found to be (almost) linearly related to the concentration. It has been asserted that the magnitude of the coefficient of proportionality, denoted B, depends on how the ions perturb the water structure. Fabio et al. use neutron scattering and simulations to look for some structural parameter that can be correlated with B. They find that the nature of the (univalent) ions is all but irrelevant to the size of the percolating water clusters in solution. The change in viscosity seems to be unrelated to any structural changes in the bulk liquid, but instead pertain to changes in the local hydration shells of the ions. As a result, they say, “the particular effect of solutes ranked in the Hofmeister series must be looked at in terms of specific ion interactions with hydrophilic or hydrophobic surfaces”, and not in terms of any generalized propensity for structure-making or –breaking.

In an intriguing preprint, Jampa Maruthi Pradeep Kanth and Ramesh Anishetty at the Institute of Mathematical Sciences in Chennai propose that the hydrophobic interaction should be understood as an effect analogous to the Casimir effect (the attraction of two surfaces separated by a vacuum due to the suppression of long-wavelength electromagnetic fluctuations of the vacuum in the gap) (preprint here). Their molecular mean-field analytical method suggests that confinement alters the allowed fluctuations of the hydrogen-bond network, specifically the long-ranged correlations between water molecular orientations. Now what I’d like to know is whether an explicit connection can be made to the alleged role of fluctuations in the Lum-Chandler-Weeks model. But that seems to argue in the opposite direction, namely that fluctuations are actually enhanced in the gap owing to the destabilizing influence of the hydrophobic surfaces on the intervening water layer. It’s not clear to me whether in Kanth and Anishetty the water in the gap is, aside from the suppression of fluctuations, any different from bulk water, except perhaps for the monolayer adjacent to the surfaces…? And why would this not work for hydrophilic confinement too?

Alenka Luzar and her colleagues Christopher Daub and Dusan Bratko have just published a review of how electric fields at interfaces can modify their wettability (Top. Curr. Chem. 10.1007/128_2011_188; 2011 – paper here). Effects of this nature may play a role in the behaviour of voltage-sensitive ion channels.

Still more on denaturants. Thomas Record and colleagues at Wisconsin ask why urea is a denaturant while glycine betaine is a protein stabilizer (E. J. Guinn et al., PNAS 108, 16932-16937; 2011 – paper here). They use osmomentry and solubility measurements to look at the interactions of these molecules with 45 model proteins, and conclude that the explanation for the different behaviours lies with the details of how and where the molecules interact with the peptide surfaces. For example, urea accumulates at amide O groups, and to a lesser extent at aliphatic carbon atoms, whereas glycine betaine is excluded from them. This adds further weight to the notion that such osmolytes exert their effects via direct interactions with proteins rather than any generalized influence on ‘water structure’.

Phosphate groups turn out to be a sensitive probe of electric fields, including those that can be induced by hydration. As Steven Boxer and colleagues at Stanford show (N. M. Levinson et al., JACS 133, 13236; 2011 – paper here), electric fields perturb the vibrational spectra of organophosphates in a way that can reveal changes in hydration within partially hydrated environments, such as the active sites of enzymes.

Is there a liquid-liquid transition in confined water? That question is investigated via MD simulations by Limei Xu and Valeria Molinero at Utah (J. Phys. Chem. B jp205045k – paper here). The possibility has been raised by simulations of water in slit-like pores 2.4 nm wide (Brovchenko & Oleinikova, J. Chem. Phys. 126, 214701; 2007). Valeria and Limei use the mW water model to look at water’s behaviour in 1.5-nm hydrophilic pores at a range of temperatures and pressures up to 4000 atm. They find that at high pressures there is a signature of a somewhat abrupt but nonetheless continuous phase transition in the supercooled regime which could be interpreted as a ‘shadow’ of a L-L transition in the bulk phase which cannot itself be accessed.

A new and unusual view of the protein dynamical transition at c.200-220 K is presented in a preprint by Andrei Krokhotin and Antti Niemi at Uppsala University (paper here). They say that it this transition can be regarded as an analogue of the transition of a high-temperature superconductor to a non-superconducting pseudo-gap state. In other words, proteins can be assigned an order parameter formally equivalent to the quasiparticle wave function of superconductors. It’s not clear to me how/if this description modifies what is known already about the transition (on which, and on the role that hydration plays, there seems still to be no real consensus), but it is an original idea.

How hydration forces assemble protein aggregates

Dielectric spectroscopy has been widely used to study the dynamics of proteins and their hydration shells, but is somewhat hampered by ambiguities about the origin of the atomistic motions that contribute to the signals. Sheila Khodadadi at NIST and colleagues have now shown just how fraught a business this is. The dielectric response of hydrated proteins typically contains two components – a fast (20-50 ps) relaxation, thought to be due to hydration water (which is slowed down relative to the bulk), and a slow (0.5-10 ns) relaxation ascribed to ‘tightly bound water’. Khodadadi and colleagues have previously questioned the former assignment (J. Phys. Chem. B 112, 14273; 2008), and now they question the latter too (J. Phys. Chem. B 115, 6222; 2011 – paper here). They say that comparisons of MD simulations to dielectric spectroscopic data suggest that the nanosecond relaxation is in fact due to motions of the protein atoms themselves, while the hydration water relaxes much more quickly.

Cytochrome c oxidase (CcO) is one of the classic examples of an enzyme that uses embedded water wires for proton transport. But how does it avoid back transport of protons along the same route? One proposal is that the water wire becomes reoriented to prevent this. Another is that a side-chain element of the hydrogen-bonded network, Glu-286, rotates to break the chain. Shuo Yang and Qiang Cui of the University of Wisconsin now show that neither process seems to be a viable gating mechanism (Biophys. J. 101, 61; 2011 – paper here). They suspect that the vectorial proton transport is probably achieved instead in a more subtle way, by stabilization of the proton-transfer transition state by the charge distribution around the active site, to explore which will require sophisticated quantum-chemical modelling.

Bacteriorhodopsin is of course another archetype for the functional water wire. It has been claimed by Klaus Gerwert at Bochum and others that the proton pumped through the protein by light absorption is stored in a water cluster inside the channel. This idea is challenged in another paper by Cui and coworkers, soon to appear in JACS (P. Goyal et al., JACS ja201568s – paper here). They say that for the proton to be stored on water requires an implausibly high increase in the pKa of a hydronium, and argue that the stored proton is instead shared between Glu194 and Glu204. One can be sure this is not going to be the last word on the matter…

I missed an interesting paper last year by Ariel Fernández and colleagues on how proteins can ‘seal’ backbone hydrogen bonds (BHBs) at their surface against competition from hydration water by a close control of the local curvature: if the BHBs are in a sufficiently highly curved location, water cannot penetrate without compromising its own H-bonded network for purely geometric reasons (E. Schultz et al., PLoS ONE 5, e12844; 2010 – paper here). Misfolded proteins fail adequately to protect their BHBs this way. This work is rather closely related to the recent paper by Fernández and Michael Lynch at Indiana on how BHBs that are ‘poorly wrapped’ by hydrophobic groups can become sites of protein-protein aggregation, which lead to complexification of the ‘interactome’ due to the degradation of wrapping created by genetic random drift (Nature 474, 502; 2011 - paper here). I have discussed that work in the August issue of Chemistry World (here – requires a subscription).

Somewhat relevant to this work is a study of protein ‘hot spots’ where preferential binding of organic small-molecule probes bind, by Frank Guarnieri of Virginia Commonwealth University in Richmond and colleagues (J. L. Kulp III et al., JACS 133, 10740; 2011 – paper here). They use simulated annealing to look at the interactions of eight probe molecules, and water, with hen egg lysozyme, and find that all the probes bind in the known binding site, which is evidently the ‘hot spot’. This implies that ligand binding isn’t simply a case of molecular recognition – the ligand is already guided towards that site by the fact that other potential binding spots are ‘guarded’ by tightly bound water. The researchers say that these hot spots can therefore also act as sites for potential protein-protein binding.

Coming back to the previous paper, Ariel also believes that poor wrapping of BHBs is implicated in the aggregation of proteins in amyloid diseases – and that these are thus potentially an alarming result of evolution’s failure to deal with random drift in anything more than an ad hoc manner. The question of how hydration is related to amyloid aggregation is also considered in a preprint by Dave Thirumalai and colleagues (arxiv 1107.4820 – paper here). They argue that “water controls the self-assembly of higher-order structures”, just as the expulsion of water is a key stage in the interaction between peptides forming beta-sheet protofilaments. Specifically, water accelerates the formation of fibrils from mainly hydrophobic peptides, but slows down the aggregation of hydrophobic sequences by stabilizing them. In the higher-order hydrophobic structures, trapped water can be considered to be analogous to water confined within carbon nanotubes – in which it seems that there is a sensitive dependence of the water structure on the width of the confining region. Thus the work nicely connects studies of confined water to an important biological problem.

A different picture – I’ve not quite figured out if it competes with or complements the above picture – of self-assembly of protein fibrils is presented by Krishnakumar Ravikumar and Wonmuk Hwang at Texas A&M (JACS 133, 11766; 2011 – paper here). They compare the role of hydration forces in the self-assembly of hydrated, dry nonpolar and dry polar surfaces, represented by collagen and two amyloidic peptides, the former a ‘steric zipper’ with interdigitating hydrophobic side-chain groups. In all cases the surfaces have hydration shells, and an oscillating hydration force as the surfaces come together due to coalescence and depletion of the hydration shells. What differs is the magnitude of these. Thus the interactions have a common origin, being water-mediated in all cases. I can’t improve on what the authors say here about the role of the hydration shells: “Thus, designating a protein surface as either “hydrophobic” or “hydrophilic” may be too simplistic of a dichotomy, as surfaces in reality lie between these idealized limits. There should be no fundamental difference in the way hydration forces arise among different types of protein surfaces, even with varying affinity for water.”

The idea that water can support two liquid states in the high-pressure supercooled regime has not been directly verified experimentally, but has received enough indirect support from both experiments and simulations to have gained wide acceptance. So a preprint from David Limmer and David Chandler at Berkeley making strong claims for the non-existence of the two liquid states is bound to cause waves (arxiv 1107.0337 – paper here). On the basis of calculations using Valeria Molinero’s recently reported mW model of water, they assert that the signatures of a phase transition seen by others relate to a solid-liquid transition, or artifacts due to imperfect equilibration (which is extremely slow in this region for, e.g. ST2 water), and that there is no sign of two liquid basins anywhere in this part of the free-energy landscape. This work, and the topic in general, will be debated in a water mini-session at the Mini Stat Mech meeting at Berkeley in January (see here), which promises to be lively.

Majed Chergui at EPFL in Lausanne and colleagues have described a nice study of the time-resolved ultrafast changes in hydration of an iodide ion as it is transformed to neutral iodine by electron abstraction, probed using XAS (V.-T. Pham et al., JACS ja203882y – paper here). This transformation changes the solute from hydrophilic to hydrophobic, and so entails a considerable reorganization of the hydration shell. The hydrogens are reoriented from pointing towards the solute to pointing away from it, forming a hydrogen-bonded ‘hydrophobic cavity’. This reorganization process takes about 3-4 ps.

Achim Müller at Bielefeld and colleagues have developed a new way to study hydrophobic species encapsulated in genuinely hydrophobic cavities. They make hollow capsules from molybdenum-based polyoxometalates, with hydrophobic interior surfaces due to ligands coordinated there (C. Schäffer et al., Chem. Eur. J. 10.1002/chem.201101454 – paper here). Much less water is found inside these shells than in those with hydrophilic interior surfaces. And hydrophobic entities such as n-hexanol are spontaneously taken up into the porous shells.

Gaurav Chopra and Michael Levitt at Stanford have used state-of-the-art quantum chemical methods to map out the hydration shell of C60 (PNAS 10.1073/pnas.1110626108 – paper here). These reveal dramatic ordering of the surrounding water, evident in the time-averaged azimuthal distribution function, which is not seen when empirical force fields are used for the calculation. Moreover, ordering of the water molecules is evident as far out as about 1 nm from the C60’s centre (as I recall, the molecule is itself about 4 Å in radius). Significantly, the azimuthal water ordering around the buckyball is not due to some kind of ice-like clathration of a non-polar species but to strong dispersion attractions between the solute and solvent molecules themselves. I guess one thing this study illustrates is the sensitivity of the important details of the hydration structure of hydrophobes to the interaction potentials used.

I think I somehow failed previously to comment on an important and provocative paper by Roy Daniel and colleagues published towards the end of last year (M. Lopez et al., Biophys. J. 99, L62; 2010 – paper here). They report significant catalytic activity of pig liver esterase in near-anhydrous conditions – not merely in a non-aqueous medium, but in a ‘dry’ powder with just 3±2 water molecules per enzyme molecule. Let me quote from the abstract: “This indicates that neither hydration water nor fast anharmonic dynamics are required for catalysis by this enzyme, implying that one of the biological requirements of water may lie with its role as a diffusion medium rather than any of its more specific properties.” They admit, naturally, that it remains an open question whether this result can be generalized to all enzymes – this one might just happen to be particularly rigid. All the same, this is most definitely a finding to chew on.

Jeremy England’s paper on allostery and hydrophobic burial, mentioned in an earlier post, has now been published in Structure (paper here). To save you a click, here’s what I said before:
The paper shows that it is possible to estimate low-energy conformational changes in a protein, such as those involved in allosteric effects, on the basis simply of residue-by-residue hydrophobic effects. Specifically, he develops a method for determining the most energetically favourable way of burying hydrophobic residues, given a particular amino-acid sequence. This amounts to identifying the particular ‘burial modes’ of any given sequence. Thus, although the stabilities of conformations are doubtless multifactorial, hydrophobicity seems to be the major governing factor.