Water in tight places

A clutch of studies this week on water in confined geometries. Alan Soper and his coworkers at ISIS have used neutron scattering to look at the structure of water within the hydropholic pores of Vycor glass, the walls of which are lined with surface OH groups [J. Phys. Chem. B 111, 5610 (2007)]. For pores 40 angstroms wide – several dozen molecular diameters – they find significant disruption of the bulk liquid structure. The average number of hydrogen bonds per molecule is decreased from about 3.6 to about 2.2, and they see structural changes extending at least two layers into the liquid owing to the orientational effects of hydrogen-bonding to surface OH. It’s perhaps a little surprising that the effect is so big, and certainly raises questions about how bulk-like cell water is (the average distance between macromolecules in the cytoplasm is just 1-2 nm).

Sow-Hsin Chen at MIT and his colleagues have continued to study deeply supercooled water. Confined in mesoporous silica with pore diameters of 15 angstroms, it can be supercooled to at least 160 K. Deeply supercooled water is predicted to have a density minimum around 70 K below the well-known density maximum at 277 K – an echo of the ice-like low-density liquid phase predicted under pressure, and of the low-density amorphous ice that has been well established already. Using SANS, Chen and colleagues now see this density minimum at around 210 K for D2O [PNAS early edition, www.pnas.org/cgi/doi/10.1073/pnas.0701352104].

In a preprint [arxiv:0705.2348], Simone Melchionna use MD simulations to look at water in hydrophobic pores. They find spontaneous cavity formation for pore diameters of around 2 nm, which they relate to previous predictions and reports of density depletion and drying at hydrophobic surfaces, particularly the Lum-Chandler-Weeks model. In narrower pores (around 1.5 nm or so), this cavitation can result in complete (but intermittent) emptying, as has been proposed in protein ion or water channels as a gating mechanism. (In those cases, the precise nature of the residues in the pore neck seems to be rather crucial.)

Finally, something with a real biomolecule in it. Gerhard Hummer and colleagues have conducted MD simulations of water within the nonpolar cavities of tetrabrachion, a protein of the hyperthermophilic archaebacterium Staphylothermus marinus [JACS asap, doi:10.1021/ja070456h]. This contains several large hydrophobic cavities linked by a central channel, and the crystal structure shows that all contain water. The researchers find that the largest cavity contains 7-9 water molecules both at room temperature and at 365 K, the organism’s optimal growth temperature. But it empties at a slightly higher temperature, around 384 K. The second-largest cavity is filled with five waters at room temperature, as the X-ray structure confirms, but this breaks up at 365 K. Thus, both cavities are close to emptying at 365 K, and Hummer et al. suggest that this emptying might create sockets into which the proteases, which bind to it in its functional state, can plug.

More Hofmeister head-scratching

A paper by Dér et al. [J. Phys. Chem. B, doi:10.1021/jp066206p] makes the bold claim of providing a general microscopic interpretation of Hofmeister effects – the ion-specific salting-in or salting-out of proteins. I’m not sure that it succeeds. The basic idea is that the ions induce changes to the protein-water interfacial tension: so-called kosmotropes make the interface more hydrophobic, and chaotropes make it more hydrophilic. I can’t help feeling that the paper is hampered from the outset by an insistence on retaining the chaotrope/kosmotrope terminology, which was coined to suggest that the respective ions ‘break’ or ‘make’ ‘water structure’. There’s no evidence that ions do either, at least in terms of exerting any global influence on the hydrogen-bonded network. Indeed, Dér et al. acknowledge that spectroscopic studies [Omta et al., Science 301, 347; 2003] show no change in hydrogen-bonding on addition of ions beyond their first hydration shell. As far as I can make out, they seem to say that preferential segregation of ions at or away from the interface means that localized effects on H-bonding can be specifically felt there. But it’s not clear to me what they are thinking of in alluding to this surface segregation of ions – there’s no reference, for example, to the studies of that (at the air-water interface) by Jungwirth, Saykally and others. Nor do the authors seem to take into account how this picture applies to hydrophobic surfaces (Bruce Berne has studied this, and found ion-specific segregation). In any event, what results seems rather unsatisfactory, since we are then left with the confusing chao/kosmotrope terminology but a hint that in fact all the action is taking place at the interface (which is probably the case) – and an attempt to explain that action in terms of macroscopic interfacial tensions (which are not known anyway for protein-water interfaces). I can’t help thinking that it remains more useful to think more explicitly about how ions might modify the nature of the microscopic protein-water interface, and how this picture changes when two surfaces come together (so that, say, adsorbed ions are excluded).

Greg Voth’s group has just published a very nice review on proton transport in aqueous systems, including all the work on ‘water wires’ in aquaporin, M2, cytochromes and other proteins [J. Phys. Chem. B 111, 4300-4314; 2007].

Sylvia McLain and colleagues have conducted an extensive neutron-scattering study of the structure of proline solutions [J. Phys. Chem. B 111, 45568-4580; 2007]. Proline acts as an osmolyte or osmoprotectant, apparently protecting proteins against denaturation under water stress. It has been suggested that this is somehow due to proline clustering in solution, but McLain et al. find no strong evidence of that. Indeed, proline seems barely to perturb water’s hydrogen-bonded network at all, while remaining sufficiently hydrated to attain good solubility. They speculate that, despite the only weak tendency of prolines to cluster, they might form a protective sheath around proteins, chaperoning them to prevent denaturation.
Update: McLain and colleagues have just published a comparison of these experimental results with computer simulations (which show reasonable agreement): J. Phys. Chem. B 111, 8210; 2007.

There's a whole lot of simulating going on

Another biological ‘water wire’ is reported by Guillaume Lamoureux at U. Penn. and colleagues [Biophys. J. doi:10.1529/biophysj.106.102756]. They have simulated that ammonium transporter AmtB of E. coli, which has a hydrophobic channel that is thought to pass ammonia but to exclude water and charged species. The simulations, however, show that water can get inside and form a three-molecule chain. How this affects the permeation of ammonia (and exclusion of ammonium) isn’t yet clear, but it appears that the mechanisms proposed so far may run into problems.

Ulf Ryde at Lund and colleagues have simulated water in the active-site cavities of four human cytochromes: P450, 2A6, 2C8 and 3A4 [Rydberg et al., J. Phys. Chem. B, doi:10.1021/jp070390c]. In contrast to the crystal structures, they find that all the cavities are filled with water. That in 2A6 is small and contains only two waters, but the others have big cavities with around 40-60 water molecules – a volume of 1500-2100 angstroms. In these big cavities, water is rapidly exchanged with the environment through three to six channels. Those in 2A6 remain bound there, although quite mobile.

L-alanine acts rather like a surfactant in a water droplet, according to the density-functional MD simulations of Ivan Degtyarenko at Helsinki University of Technology and colleagues [J. Phys. Chem. B 111, 4227; 2007]. The amino acid moves to the droplet surface with its methyl group exposed, and the primary hydration shell of (on average seven) ordered and rather rigid water molecules forms around the carboxylate and ammonium groups.

More on the hydration of urea comes from Hinonori Kokubo and Montgomery Pettitt [J. Phys. Chem. B doi:10.1021/jp067659x]. They suggest that, to put it crudely, urea passes almost unnoticed in water – certainly, there’s no evidence of it acting as a structure-breaker. Another nail in the coffin for this hoary old idea.

Acid on top

[Victoria Buch and coworkers have just published a paper in PNAS on the acidity of the water surface, which is likely to have biochemical consequences. Here is the pre-edited article that I’ve written on the result for the May issue of Chemistry World.]

Pure water has an acid skin. This striking notion has been confirmed by calculations and tests by an international team of scientists. Victoria Buch of the Hebrew University of Jerusalem in Israel and her coworkers have found that the pH of pure water falls from the perfectly neutral value of 7 within the liquid to just 4.8 or less – about as acidic as beer – where water meets air at the surface [V. Buch et al. PNAS online, doi:10.1073/pnas.0611285104].

The finding could be significant for a number of disciplines. In the atmosphere, many important chemical reactions between trace gases take place at the surface of water droplets in clouds. “pH is an essential factor for many of these reactions”, says Pavel Jungwirth, one of Buch’s collaborators at the Academy of Sciences of the Czech Republic in Prague. “The low pH could also affect the rates of carbon dioxide absorption at the ocean surface”, adds water specialist Richard Saykally of the University of California at Berkeley.

And in molecular biology the effect might be reproduced where water comes into contact with water-repelling (hydrophobic) parts of proteins, changing the acid-base chemistry that goes on at protein surfaces. “The effects of interface acidification on protein and membrane structure could be huge”, says theoretical chemist Gregory Voth at the University of Utah.

“I think that this is a very important paper”, says Saykally, who has previously found indirect evidence of the surface acidification. While the prediction was well-known, he says, “this is the first study to actually predict the pH of the liquid water surface.”

It has become clear in the past several years that the water surface may look very different from the bulk liquid. Saykally and Jungwirth have shown that dissolved ions can become either depleted or concentrated at the surface [e.g. P. B. Petersen & R. J. Saykally, Annu. Rev. Phys. Chem. 57, 333; 2006]. But the accumulation of protonated water molecules (H3O+ or hydronium, the basic component of acidity in water) at the air-water interface happens for unique reasons.

In 2004 Voth and his coworkers used quantum-mechanical computer simulations to show that hydronium prefers to sit at the water surface rather than deep inside the liquid [M. K. Petersen et al. J. Phys. Chem. B 108, 14804; 2004]. Whereas H2O molecules typically form four hydrogen bonds to their neighbours – two via the hydrogen atoms, and two via the electron lone pairs on oxygen – H3O+ can only form three. The three hydrogens can bind to water molecules, but the oxygen atom, where most of the positive charge resides, can’t any longer act as a good ‘acceptor’ for hydrogen bonds.

This makes it energetically unfavourable for the oxygen side of hydronium to be in water at all. So, Voth’s team said, hydronium acts somewhat like an amphiphile – a molecule with a water-soluble part and a hydrophobic part, like a soap molecule. The ions gather at the air-water interface with the hydrogens pointing downwards to make hydrogen bonds and the oxygen pointing up out of the liquid [S. S. Iyengar et al. Int. J. Mass Spectr. 241, 197; 2005].

In 2005 Saykally and his colleague Poul Petersen used a spectroscopic technique to adduce indirect evidence for this surface excess of acidic hydronium [P. B. Petersen & R. J. Saykally, J. Phys. Chem. B 109, 7976; 2005]. Buch and colleagues now have fresh evidence, albeit still somewhat indirect: they show that deuterated water D2O can swap hydrogens with H2O about 20 times faster at the surface of ice nanocrystals (which have a liquid-like surface) than deeper inside. This switch depends on the presence of hydronium ions.

The researchers also have new simulation data for the surface acidification, which enables them to estimate the pH increase. But Voth cautions against placing too much weight on this number as yet, because of the simplifications in the model. For example, “they have no possibility of having the hydronium and hydroxide recombine into a water molecule”, he says.

A key question now is whether the experimental evidence could be made more direct – “ice crystals are not that relevant to water.” says Voth. But this will be a challenge: “it is very hard to measure the top surface layer of a volatile liquid like water”, says Jungwirth. Saykally agrees with that. “We are thinking hard about how to do this,” he says, “but it’s not easy.”

Smooth folding

A paper in the forthcoming issue of PNAS (104, 6206; doi/10.1073/pnas.0605859104) by Peter Tieleman’s group at the University of Calgary looks at how to reconcile the energy-funnel picture of protein folding with the fact that there are likely to be significant enthalpic barriers to the folding process. They use simulations of the association of two polyalanine and two polyleucine alpha-helices to figure out whether enthalpic barriers exist (they do), and why. It seems they arise in this case from the fact that, as the chains approach, they must become desolvated before there is a compensating enthalpic gain from strong helix-helix interaction. (Interestingly, the researchers see no dewetting transition as the helices approach, of the sort predicted by Lum, Chandler and Weeks (J. Phys. Chem. B 103, 4570-4577; 1999), but only ‘steric dewetting’ when there is simply no longer space to fit in a layer of water. This contrasts with the simulations of hydrophobic plates by Bruce Berne mentioned in my previous post (JACS asap doi:10.1021/ja068305m), where dewetting does feature. It is possible that this might be rather sensitive to the precise geometry, size and hydrophobicity of the two surfaces.) The enthalpic energy barrier is, however, largely compensated by the gain in solvent entropy on desolvation, leading to a free-energy barrier that is very small (for poly-A) or non-existent (for poly-L). Thus, the idea of a relatively smooth free-energy funnel is recovered.

I’ve now had a better look at the Berne paper. It suggests that Hofmeister effects are indeed complicated, and not best explained by a simplistic structure-making/breaking model. In the case of the association of nanoscale hydrophobic surfaces, the effect of ions depends on whether or not they accumulate preferentially at the surfaces. High-charge-density ions induce salting-out (reducing the solubility of hydrophobes) via an entropic effect due to preferential exclusion of ions from the interfaces. Medium-charge-density ions induce salting in because of a different entropic effect, due to strong hydration of the ions and a consequent reduction in solvent entropy when the ions, preferentially adsorbed at the surfaces, are expelled as the surfaces associate (I think I’ve got that right). But low-charge-density ions cause salting in enthalpically, since they bind to the surfaces and lower the surface tension of the plate-water interface, thus lowering the enthalpy of association. As if this isn’t complicated enough, Berne and colleagues say that something quite different applied for electrolytes and small hydrophobic particles (see Zangi & Berne, J. Phys. Chem. B 110, 22736-22741; 2006). Hmm.

Not strictly related to biochemistry, but Rudy Marcus and Yousung Jung have just published a proposed explanation for why there is a rate acceleration of certain organic reactions, particularly those associated with ‘click chemistry’, at the interface of water and an organic phase: see JACS asap doi:10.1021/ja068120f.

Why cell fluid is lumpy

How homogeneous is the cytoplasm? There is increasing evidence that proteins in concentrated solution form relatively long-lived clusters. Wilson Poon and his colleagues showed in 2004 that this effect, previously rather anecdotal, is real and general, applying to colloidal particles as well as proteins (Stradner et al., Nature 432, 492; 2004). They showed using small-angle X-ray and neutron scattering that lysozyme forms clusters of about 3-10 molecules at volume fractions of between 0.05 and 0.2, which they say are equilibrium structures resulting from the interplay of short-ranged attractive (van de Waals) and electrostatic repulsive forces. Now Peter Vekilov at Houston and his coworkers broadly support that notion by looking at concentrated solution of bacterial lumazine synthase using light scattering (Gliko et al., J. Phys. Chem. B 111, 3106; 2007). They see clusters with lifetimes of around 10 s and a mean radius of about 350 nm (individual molecules are bout 15.6 nm in diameter). Changing the protein concentration changes the cluster concentration (which can reach a volume fraction of 0.001), but not the cluster size. But Velikov et al. say that cluster formation and size is dominated by kinetics, not thermodynamics: these clusters are metastable with respect to both protein crystals (i.e. in supersaturated solution) and to well-dispersed solution.

All of this recalls the flurry of interest in clustering excited by the work of Geckeler and Samal on C60 (Chem. Comm. 2001, 2224), which was rather breathlessly touted as a possible mechanism for homeopathy. That work was truly odd, as it seemed to suggest that the cluster size increased with increasing dilution. I’m not aware that the result has been reproduced. Needless to say, the homeopathy connection makes no sense (at best, you get a few ‘active’ bottles and the vast majority containing just water); but at the very least, there’s no reason to regard clustering per se as perplexing or odd.

The question of a vapour gap at the interface of water and a hydrophobic surface, and its relation to the long-ranged hydrophobic attraction, seems to be resolving itself. It seems now that a very thin depletion layer exists. But the role of dissolved gases in forming nanobubbles remains to be fully resolved (see ‘Why does water do that’ below). They have been seen by various methods, but with the proviso that they could possibly be an artefact of the probe technique, and that they haven’t been shown definitively to be gaseous anyway. Also, small nanobubbles should have a small radius of curvature and thus a large Laplace pressure, promoting their dissolution. William Ducker and colleagues at the University of Melbourne have now shown that flat gas bubbles, about 5-80 nm thick and 4 microns across, can exist at such a hydrophobic interface for over an hour (Phys. Rev. Lett. 98, 136101; 2007). This size means that the internal pressure is barely above atmospheric. But the bubbles form only when a particular protocol is followed for introducing the gas layer (carbon dioxide): in other words, “the presence of the gas phase depends on the previous history of the interface.”

Ivan Brovchenko and colleagues recently linked the low-hydration polymorphic transitions of B-DNA to the presence (or not) of a fully connected (percolating) network of water molecules in the hydration sphere (Brovchenko et al., Phys. Rev. Lett. 97, 137801; 2006). They’ve now extended that work by looking at the percolation transition for both B- and A-DNA (Brovchenko et al., J. Phys. Chem. B 111, 3258; 2007). Although the percolation thresholds (that is, the surface coverage of water on the DNA molecules) are virtually identical, the mechanisms are quite different in each case: the threshold corresponds to the appearance of a spanning water network in the major groove of B-DNA, but the minor groove of A-DNA. It isn’t clear, then, whether the near-coincidence of the two thresholds is indeed just coincidence or has some deeper physical cause. In any event, there are also insights here into how ions can alter the hydrogen-bonding patterns and thus shift the thresholds.

Water seems to play an important role in electron transfer between some protein redox centres – this was discussed nicely by Gray & Winkler recently (PNAS 102, 3534; 2005). They enumerated the various ways, direct and indirect, that water might facilitate electron hopping. Agostino Migliore and colleagues in Modena have now used ab initio calculations for the copper active sites of azurin to figure out how water-mediated pathways are functioning in this case (Migliore et al., J. Phys. Chem. B, advance online publication, doi:10.1021/jp068773i). But it’s a complicated picture that emerges, in which no one physical pathway seems to be responsible for what is observed. Sorry, but it’s hard to put this one into more of a nutshell than that.

Finally, and perhaps even more cryptically, I want to flag up a paper by Bruce Berne and colleagues in JACS ASAP (doi:10.1021/ja068305m) on the effect of ions on the hydrophobic interaction between two plates. This complements Berne’s recent study of much the same thing for hydrophobic particles (J. Phys. Chem. B 110, 22736; 2006). The phenomenon is of course intimately related to salting-in/out and Hofmeister effects, and as such, contains a lot of important information on the effect of electrolytes on protein aggregation and folding. So there’s a lot of good stuff here to digest, and I’m not going to manage that in a hurry without risking indigestion (or more probably, misapprehension). Worth spending time on.

How thick-skinned is hydration?

How much water do you need to fully solvate a protein? There are many studies of protein behaviour at low water coverage, going back to the suggestion by Rupley and Careri (Adv. Protein Chem. 41, 37-172; 1991) that proteins seem to require about 0.4 g of water per gram of protein to achieve their normal functionality. Roland Winter and coworkers have investigated the notion of a percolation transition in water coverage that brings the protein dynamics to life (Oleinikova et al., J. Phys. Chem. B 109, 1988-1998; 2005; Smolin et al., J Phys. Chem. B 109, 10995-11005; 2005). Mehdi Bagheri Hamaneh and Matthias Buck at Case Western have looked at the question in a rather different light: how much water do you need to put around a protein in order to be able to simulate it realistically? They find (Biophys. J. 92, L49; 2007) that you don't need to fill up your simulation box with explicit water – a shell just two or three layers thick (using the CHARMM22/CAMP potential function) will do the job well enough. That's computationally cheap, and I suppose implies that there's not really much excuse for failing to model hydration explicitly. It also implies that the celebrated cooperativity of water dynamics in the hydration shell does not appear to extend very far – at least, perhaps one should say, for the case of lysozyme considered here.

More on the 'glass transition' at around 220 K, seemingly shown now (Chen et al., PNAS 103, 901; 2006) to be a fragile-to-strong crossover. This applies also to DNA (Chen et al., J. Chem. Phys. 125, 171103; 2006), and now Sow-Hsin Chen at MIT and coworkers have found similar behaviour for RNA, again at 220 K (http://xxx.arxiv.org/abs/physics/0703166). So this seems to be pretty universal behaviour for biomacromolecules, reinforcing the idea that the change in dynamics is imposed by the hydration water.

Masahiro Kinoshita has sent me a couple of his papers from Chem. Phys. Lett. (see them here and here) which explore his idea that "the major driving force in protein folding is a gain in water entropy". In a nutshell, they say that "a protein is designed to fold into the structure that maximizes the entropy of water under the requirement that sufficiently many intramolecular hydrogen bonds be formed to compensate the dehydration penalty." In other words, as I understand it, the enthalpies balance and what's left (governing stability) is the water entropy change. That's intriguing; I'm still struggling to see how this ties up with Jack Dunitz's suggestion (Science 264, 670; 1994; Chem. Biol. 2, 709-712; 1995) that transferring a water molecule from an ordered binding site where it is bound by an ‘average’ hydrogen bond to the bulk involves an overall free-energy change that is close to zero, and with ideas about the importance of interactions at specific residues – the 'hotspots' discussed in the last post, and Ariel Fernandez's notion of dehydrons, for instance. One day, perhaps, it will all make sense to me.