Thursday, March 19, 2015

JCP special issue on biological water

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

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



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

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

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

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

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

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



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

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



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



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

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

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

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

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

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

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

Monday, December 8, 2014

What to think about Kauzmann

I guess you can tell when I’m on travel, because that is when so many of these posts tend to get done – on this occasion, on a visit to the rather wonderful Chemical Heritage Foundation in Philadelphia (and yes, had I had more time then I should surely have liked to do some more visiting in the locale, since I know some readers were nearby!).

“Restoring Kauzmann’s 1959 explanation of how the hydrophobic factor drives protein folding” is quite a big claim. But what Robert Baldwin at Stanford means by making this claim in the title of his PNAS paper (PNAS 111, 13052; 2014 – paper here) is not that, as Kauzmann argued, hydration water is a kind of ordered, ice-like clathrate, but rather that the driving force of hydrophobic attraction is ultimately entropic, being due to the release of relatively constrained waters. Baldwin argues that a dynamic but relatively ordered hydration shell due primarily to van der Waals attraction can equally account for the hydration energetics: why, for example, the hydrophobic free energy depends on the solvent-accessible surface area of the nonpolar interface of hydrophobes. What I still find a little puzzling about this analysis, however, is the apparent implication that there was any doubt about the existence of non-bulk-like hydration shells around hydrophobic groups. That, surely, is clear by now; what seems less agreed is whether or not these shells have waters with retarded dynamics, stronger hydrogen-bonding and so forth.

This issue is also directly confronted in a paper by Wilbee Sasikala and Arnab Mukherjee of the Indian Institute of Science Education and Research in Pune (JPCB 118, 10553; 2014 – paper here). They calculate the translational and rotational entropy of single water molecules as a function of their distance from hydrophobic solutes and find that, intriguingly, the entropy gradually increases with distance for small (>0.746 nm) hydrophobes but that the translational entropy decreases with distance for larger hydrophobes. The rotational entropy still increases with distance in this latter case, so that the crossover for the sum of the two in fact occurs at solute sizes of around 1.5 nm – consistent with David Chandler’s suggestion of a crossover in styles of hydrophobic hydration at around this scale. The increase in translational entropy close to large hydrophobes seems to be related to the relatively larger number of dangling H-bonds in that case.

These questions are also touched on from a different direction in a paper by Robert Harris and Montgomery Pettitt (PNAS 111, 14681; 2014 – paper here) in which they examine the energetics of cavity formation for a nonpolar van der Waals solute inserted into water. Although their calculations of the free-energy perturbation for a series of alkanes fits the standard idea that the solvation free energy depends linearly on surface area (as Baldwin notes), nevertheless the contributions to this trend for each atom in the alkanes are not simply additive but depend on correlations with the neighbouring atoms. Or to put it another way, the various contributions to the free energy change can’t be calculated by assuming a constant surface tension for the cavity interface; there are thus subtle changes in the water density around the solute that a complete theory of hydrophobic hydration will need to take into account.

As described by David Chandler and his coworkers, the dewetting transition that may drive hydrophobic attraction between extended surfaces is triggered by unusually large-amplitude fluctuations. This picture has often been advanced for the case of purely hard-core hydrophobic surfaces. Richard Remsing and Amish Patel at the University of Pennsylvania have investigated whether that picture is modified for the case of realistic solutes with attractive van der Waals interactions with the solvent (http://www.arxiv.org/abs/1410.1614). They find that, when the attraction is felt in the hydration-shell alone (that is, not in the solute core), it makes essentially no difference to the water density fluctuations.

There is something of the Kauzmann spirit in the convenience of the explanation for crowding effects that explains them in terms of entropic effects: namely, that crowding agents such as glucose and PEG exert their influence via excluded-volume effects due to hard-core repulsions. Simon Ebbinghaus and colleagues at Bochum challenge this view in a paper that argues for a role of enthalpic effects too (M. Senske et al., JACS 136, 9036; 2014 – paper here). They study the thermodynamics of ubiquitin folding in the presence of cosolutes such as sugars, PEG and salt, using CD spectroscopy and DSC. They find that the temperature dependence of the heat capacity change on unfolding has an important role, which implies some enthalpic influence. They suggest that, for crowding agents like glucose and dextran, this influence might be exerted by cosolute-induced distortions of the hydrogen-bonded hydration network around the protein, i.e. it is solvent-mediated. This suggests a new framework for understanding crowding effects in terms of a balance between entropic and enthalpic contributions.

It has been suggested that water molecules trapped in internal cavities of thermophilic proteins might contribute to their enhanced thermal stability. Might they provide a hydrogen-bonded network that helps to stabilize the molecule and inhibit the formation of internal voids as an initial stage in denaturation? Fabio Sterpone at the University Paris Diderot and his coworkers investigate this question for the hyperthermophilic domain of a protein from S. solfataricus, using MD simulations and free-energy calculations to compare it with a homologous domain from an E. coli protein (O. Rahaman et al., JPCB ASAP jp507571u – paper here). Although under ambient conditions the internal hydration for the thermophilic protein is more favourable than for the mesophilic one, at the high temperatures at which the former operates the cavities are largely empty anyway. However, fluctuations in the number of buried waters appear to be intimately connected to the conformational fluctuations of the protein: the more hydrated cavities of the thermophilic protein seem to provide access to multiple conformational states, belying the common notion that such proteins are more rigid than mesophilic homologues.

[n.b. I have just come across this preprint, which, while not discussing thermophiles, is extremely relevant to the issue in its analysis of the role of internal cavities, and their hydration state, for protein conformational flexibility.]

Martina Havenith and her coworkers at Bochum have previously provided evidence from THz spectroscopy that rather long-ranged gradients in solvent dynamics may play an important role in the binding of substrates in an enzyme’s active site as it forms the transition-state complex. Now they report something even more remarkable: that coupling of solvent and protein dynamics exhibit correlations on timescales that exceed the duration of a single catalytic cycle, indicating coupling that is not accounted for within conventional Michaelis-Menten steady-state theory (J. Dielmann-gessner et al., PNAS advance online publication 10.1073/pnas.1410144111 – paper here). These couplings are substrate-specific, and they contribute to the enzyme’s reactivity. In calling water “more than a bystander”, I have to say that I had not imagined that its participation would extend so deeply. I suppose one must assume that MM kinetics remain a good approximation to what transpires in most cases, but this is a striking illustration of what a delightful collaboration of solvent, protein and substrate is entailed in the fuller picture.

Barry Sharpless’s work on “on-water” reactions – the acceleration of various organic reactions when they happen at the interface of water and an organic solvent (S. Narayan et al., Angew. Chem. Int. Ed. 44, 3275; 2005) – was extremely interesting but never fully explained. One idea was that transition states were being stabilized by dangling hydrogen bonds at the interface. Thomas Kühne at Paderborn and his coworkers have now examined this idea for the case of a Diels-Alder reaction via quantum-chemical MD simulations, and find that while the effect dos occur, it seems to be rather less significant than has often been supposed – the number of H-bonds to the transition state is only marginally increased at the interface compared to the homogeneous situation (K. Karhan et al., http://www.arxiv.org/abs/1408.5161 (2014)).

The release of a proton from photo-excited retinal in bacteriorhodopsin – the initial stage of the molecule’s photocycle – is accompanied by a twist of the retinal photoproduct. Is this twist governed by the intrinsic properties of retinal or by interactions with the protein/solvent environment? Marcus Elstner at the Karlsruhe Institute of Technology and colleagues study this question using quantum-chemical MD (T. Wolter et al., JPCB ASAP jp505818r – paper here). The answer is complex, especially in its temperature dependence, but it does seem that a twisted retinal conformer is somewhat stabilized by interactions with the protein side chains and water molecules in the active site. It seems that relaxation of the twisted chromophore back to its planar state could involve translocation of one water molecule from the extracellular to the cytoplasmic side of the complex – but that can’t yet be confirmed either way from these calculations.



There seems to be a rather more clear mechanism by which active-site water plays a functional role in the related case of rhodopsin, according to simulations by Yaoquan Tu and colleagues at the KTH Royal Institute of Technology in Stockholm (X. Sun et al., JPCB 118, 10863; 2014 – paper here). They find that a rearrangement of the hydrogen-bonded network around the Schiff base of rhodopsin, due to movement of one particular water molecule, might be responsible for the switch from the inactive to the constitutively active state, mediating proton transfer from the base to the Glu113 group.



[Proposed water-mediated mechanism for activation of rhodopsin. You won’t be able to see much from this image alone, I guess – the text of the paper explains the details indicated by the red arrows. But it’s the IW6 hydration site towards the top of the active site that is proposed as the crucial switch.]

“Is urea a structure-breaker?” is the provocative question posed by Niharendu Choudhury and colleages at the Bhabha Atomic Research Centre in Mumbai (D. Bandyopadhyay et al., JPCB 118, 11757; 2014 – paper here). You might be tempted to respond “Is that really the right question?”, but this is in a sense the researchers’ point: the considerable debate around the mechanism of urea’s denaturant properties has often been conditioned by notions of the breaking (or otherwise) of water structure – the so-called indirect effect on macromolecular structure. Yet is there any real evidence for it? Using MD simulations, Choudhury and colleagues conclude that, even at high concentrations, urea does not significantly disrupt the tetrahedral structure of water. Rather, it replaces water rather neatly in the hydrogen-bonded network. The authors admit that this does not yet really pronounce on the situation with macromolecules present, in terms of the nature and balance of solvent-solute-cosolute interactions. The question of whether any of this should be broached in terms of alleged “chaotropicity” is one to which I will return shortly…

How hydration properties affect the behaviour of intrinsically disordered proteins has become a focus of considerable attention recently. Sanjoy Bandyopadhyay at the Indian Institute of Technology in Kharagpur and colleagues have investigated this issue using MD simulations of an IDP, amyloid beta, in comparison with the globular protein ubiquitin (J. C. Jose et al., JPCB 118, 11591; 2014 – paper here). They find that the hydration water for the IDP is marginally less strongly coupled to the protein dynamics, and more bulk-like, than it is for UBQ. The water dynamics are more heterogeneous, apparently because of the conformational fluctuations of the protein. To return to the questions above, this arguably implies that there should be a smaller entropic driving force for hydrophobic association of the IDP – to put it another way, the protein’s surface is rendered relatively less hydrophobic.

The conventional view of antifreeze proteins (and glycoproteins) as acting via direct binding of ice at their surfaces was recently supplemented by the observation of long-ranged (up to 2 nm from the surface) retardation of water dynamics (S. Ebbinghaus et al., JACS 132, 12210; 2010). This picture is supported by MD simulations by Anand Narayanan Krishnamoorthy and colleagues at the University of Stuttgart (JPCB 118, 11613; 2014 – paper here). They find that hydrogen-bonding groups – hydroxyl in threonine, disaccharides – at the protein surfaces are mostly responsible not only for direct water binding but also for the long-range dynamical perturbation. Osmolytes, including chaotropes such as urea and (especially) kosmotropes such as hydroxyectoine, enhance this dynamical effect. Meanwhile, Alexander MacKerell at the University of Maryland and coworkers also find in MD simulations that carbohydrate groups on AFGPs not only engage in hydrogen-bonding with the solvent but also modify the tetrahedral arrangement and the dynamics of water molecules as far as 12Å from the surface – but only at low temperatures (<250 K) (S. S. Mallajosyula et al., JPCB 118, 11696; 2014 – paper here). They propose that the dynamical effect is in fact the dominant influence on the antifreeze behaviour.

The best way to characterize the hydrophobicity of amino acid side chains has been much debated. Timir Hajari and Nico van der Vegt at TU Darmstadt extend the emerging focus on context-dependence of the issue by computing solvation free energies for the side chains in a way that factors in the effects of the peptide backbone (JPCB 118, 13162; 2014 – paper here). They find that the backbone effects are far more significant for nonpolar than for polar side chains, in the former case reducing the hydrophobicity relative to what is found for the isolated amino acids. This might support the view that intramolecular hydrogen-bonding in the peptide is a more important driving force for protein folding than are hydrophobic interactions.

Adam Perriman, Stephen Mann and their collaborators at Bath recently described a technique for preparing solvent-free protein liquids by coating the surfaces with electrostatically bound polymer surfactants (Perriman & Mann, ACS Nano 5, 6085; 2011). Now they show that lipases prepared this way remain catalytically active despite having only 20-30 water molecules per molecule, which is of the order of 2% of what is needed to cover the solvent-accessible area (A. P. S. Brogan et al., Nature Commun. 5, 5058: 2014 – paper here). What is more, the proteins remain active up to temperatures of at least 150 C.

Huaqiang Zeng of the Institute of Bioengineering and Nanotechnology in Singapore have created synthetic molecules based on pyridine that form helical structures with a pore about 2.8Å threading through them, which they hope might mimic the water-conducting channels of aquaporins (W. Q. Ong et al., Chem. Commun. 47, 6416; 2011). Now they report that these constructs can be threaded by a water wire that permits not only proton transport but also osmotically driven water-molecule transport across lipid membranes in the presence of a proton gradient (H. Zhao et al., JACS 136, 14270; 2014 – paper here).

To what extent the state of hydrated protons is influenced by quantum effects has been quite widely studied, but Ali Hassanali at the Abdus Salam International Centre for Theoretical Physics in Trieste and coworkers revisit the question using state-of-the-art quantum chemical methods (F. Giberti et al., JCPB 118, 13226; 2014 – paper here). They find that the classic Eigen and Zundel ions still dominate, but that there can be “wild fluctuations” in which the proton is extended over long proton wires involving 2-5 water molecules. These fluctuations reduce the effective hydrophobicity of the hydrated proton.

Wednesday, November 12, 2014

Hydrophobic or not?

You thought buckyballs were the archetypal hydrophobic substance? Me too. But Li et al. have found in molecular simulations that the interaction of two C60 molecules in water has a repulsive contribution for the solvent: water actually seems to push the molecules apart (Li et al., Phys. Rev. E 71, 011502, 2005; J. Chem. Phys. 123, 204504; 2005). The same seems to be true of two carbon nanotubes when they come into close proximity with a particular alignment of their axes (Uddin et al., Polymer 52, 288; 2011; Ou et al., JPCB 116, 8154; 2012).

How can this be possible? How can buckyballs be hydrophobic and at the same time apparently attracted to waters more than the waters are attracted to themselves? Ronen Zangi has recently addressed this question using molecular dynamics simulations (J. Chem. Phys., in press). He points out that buckyballs lie right at the 1-nm crossover point predicted by David Chandler and colleagues for different modes of hydrophobic hydration. But it seems that they act more like large rather than small hydrophobes, in that it’s not possible for the water to rearrange so as to preserve the hydrogen-bonding network as it can for small hydrophobic molecules.

However, buckyballs aren’t like a pair of hydrophobic plates. They are of course curved, convex surfaces, and we know that hydrophobic solvation is sensitive to curvature (Wallqvist & Berne, JPC 99, 2885; 1995). Ronen finds that, because the actual contact area of two buckyballs is rather small, the favourable free energy change for association can’t be attributed to strong solute-solute interactions, as it is for two graphene sheets say.

So the various influences here on the free energy of association are subtle. However, the crucial point is that, as the buckyballs come together, some of the hydration water changes character. The waters in the primary hydration spheres are already somewhat compromised, having on average a smaller number of hydrogen bonds than those in the bulk (these are shown in grey below). But when the buckyballs are only a few nanometers apart, there is a new class of water molecules in between them that are even more depleted of hydrogen bonding (shown in orange). And the key point is that, unlike the case of flat plates in contact, some of these anomalous water molecules remain even when the buckyballs are in contact.



So there is a complex reckoning here of the entropic and enthalpic effects, coming from the fact that the various factors are not simply additive because of the particular scale and geometry of the hydrophobic interaction. Ronen concludes that “bucky-balls can serve as an example in which hydrophobic interaction cannot be deduced from hydrophobic solvation”. Or to put it another way, the effective pair interaction of the solute is not hydrophobic, in that solvent contributes a repulsive influence, and yet the solvation properties are hydrophobic because of the qualitative difference between the solvent-separated and fully associated particles. This, I think, is one of the best illustrations I have seen that hydrophobicity is a very slippery concept.

Martina Havenith has written a nice Perspective for JACS, with Valeria Conti Nibali, on the use of THz spectroscopy to study biomolecular hydration (JACS 136, 12800; 2014 – paper here). In particular, they discuss recent work on ligand binding and antifreeze proteins which points to the existence of a gradient in water dynamics towards the active sites, acting as a “hydration funnel”. The concept is nicely illustrated in this graphic:



You won’t be surprised to hear me cheer on the final conclusion: “So far, in all these applications, the solvent is still a strongly underestimated and mostly neglected element of the multilateral partnership in biomolecular function.”

Intrinsically disordered proteins tend to form loose but collapsed globules that trap some water. Some of these, such as kappa-casein, have charged and basically hydrophilic residues, and their collapsed conformations trap a fair amount of water. Shruti Arya and Samrat Mukhopadhyay of the Indian Institute of Science Education and Research in Mohali have studied the dynamics of this water within globules of kappa-casein using time-resolved fluorescence spectroscopy to measure the solvation time of a fluorescent dye probe (JPCB 118, 9191; 2014 – paper here). They find that the water relaxation times are three orders of magnitude slower than the bulk, and an order of magnitude slower that that typically found at protein surfaces. In other words, they say, it seems to form a highly ordered network within the disordered globule. They speculate that the entropic gain on release of this water light explain the oligomer formation that initiates the formation of amyloid fibrils from IDPs.



A similar approach of adding a dye-sensitized group (an aza-Trp) to ribonuclease T1 enables Wei-Chih Chao and colleagues of National Taiwan University to examine the water dynamics in this protein, and specifically in the connecting loop region of the molecule (W.-C. Chao et al., JPCB ASAP jp503914s – paper here). They find that the decay dynamics can be fitted with a two-component model, leading them to propose two conformational forms, which they call the loop-open and loop-close(d?) forms. Simulations support this idea and suggest that interconversion of the two conformers involves changes in the water network around the substituted Trp group, with water being squeezed out of the loop-close form. What I don’t get too clearly – my fault, I’m sure – is how/if these conformational changes relate to enzymatic function.



Domains of membrane proteins with different dynamics have different hydration dependence, according to Jun Wang and colleagues of the Wuhan Institute of Physics and Mathematics (Z. Zhang et al., JPCB 118, 9553; 2014 – paper here). They use NMR to follow the dynamics of the protein chains in diacylglycerol kinase, and find that while the highly mobile regions are highly sensitive to changes in hydration – the fast, large-amplitude motions are suppressed below 20% hydration – the dynamics of the more rigid domains are insensitive to hydration. This, I daresay, is what one might expect given that the rigid domains are those embedded in the lipid membrane while the mobile domains generally extend beyond it. But is this hydration dependence an epiphenomenon of the demands for folding and packing the respective regions, I wonder, or essential to those structural differences?

I recently wrote a feature for Chemistry World about the internal structures of quiescent cells and spores (here, but behind a paywall I fear), which looked to some extent into the question of what the state of the solvent in these cells is. That is now probed too by Charles Rice at the University of Oklahoma and colleagues, using deuterium NMR to look at water dynamics in bacterial (B. subtilis) spores (A. W. Friedline et al., JPCB 118, 8945; 2014 – paper here). They conclude that the water in the cytoplasm is in a mixture of states: there is some water that is mobile and accessible to proton exchange with the external environment, and also some that is more rigid and inaccessible to exchange. Some of the latter seems to be “bound water” associated with hydrated biomolecules, but some is sequestered in an essentially rigid core, which is however not ice-like. This raises questions of whether such water is a passive consequence of the dormancy of the cell (for example because of the relative lack of molecular motion such as that driven by transport motor proteins), or an active aspect of that shutdown, which perhaps helps to protect the molecular ingredients. And is it gel-like, glassy, or…? An interesting contribution to an unfolding story.



What do alcohols do to water? In particular, does an increasingly long aliphatic tail increasingly disrupt “water structure”? Well actually, no, according to the X-ray Raman scattering study of Iina Juurinen and colleagues at the University of Helsinki (I. Juurinen et al., JPCB 118, 8750, 2014 – paper here). They see no substantial difference in the effects on either the hydrogen-bond network of the solvent as a whole, nor in the tetrahedrality of the alcohols’ solvation water, in progressing from methanol to ethanol and 1-propanol. This supports the earlier conclusions for methanol alone by Dixit et al. (Nature 416, 829; 2002), who found that the total number of hydrogen bonds is unchanged in adding the alcohol to water.

And what drives the association of glycine oligomers in water (such systems being sometimes considered simple models of intrinsically disordered proteins)? Montgomery Pettitt at the University of Texas in Galveston and colleagues explore that issue through simulation of Gly5 (D. Karadur et al., JPCB 118, 9565; 2014 – paper here). They conclude that the aggregation is not driven by hydrogen-bonding but by electrostatic interactions between the partially charged atoms. Gly5 can’t really be considered hydrophobic, so hydrophobic interactions don’t obviously play a part, although interestingly the researchers see some features often associated with them.

Some brief glimpses. Harsha Annapureddy and Liem Dang at PNNL in Washington present a summary of their attempts to understand water exchange in the solvation shells of ions using molecular simulations, for example by calculating the potentials of mean force (JPCB 118, 8917; 2014 – paper here). Giuseppe Bellavia and colleagues at the University of Lille explore how glycerol further enhances the denaturant properties of trehalose by rigidifying the (still liquid) solvent matrix (G. Bellavia et al., JPCB 118, 8928; 2014 – paper here). Timothy Duignan and colleages at ANU calculate ion solvation energies at the air-water interface and say that their findings can reproduce the surface tensions of electrolyte solutions (T. Duignan et al., JPCB 118, 8700; 2014 – paper here). And on a similar Hofmeister theme, Ferenc Bogár at the University of Szeged and coworkers report MD simulations of the effect of various salts on the interfacial tension between water and a small model protein (F. Bogár et al., JPCB 118, 8496; 2014 - paper here).

OK, with apologies to those of you who have sent me papers, I will leave it here for now and deal with them in the next post – and I’m still only caught up to early August… Oh, and I should just add that I thoroughly enjoyed this recent conference on nanobubbles in Shanghai, hosted by the Shanghai Institute of Applied Physics. And that offers me an excuse for a parting remark from Confucius that some of you might find reassuring: “the intelligent find joy in water.”

Monday, September 1, 2014

Life after Gordon

From time to time I wonder to myself if the number of folks reading this blog can be counted on the fingers of one hand – but judging from the kind comments I received at the wonderful Water Gordon Conference in July, I would need at least my toes too. More importantly, it seems to be appreciated; Shekhar Garde was even kind enough to advertise it in the New York Times, which makes me smile somewhat at the bemusement it might have elicited in some NYT readers who perhaps tried it out. In any event, you have persuaded me to keep it up; indeed, to begin the next post on the flight home [but not to finish it then, I fear…]

I spoke at the meeting about some of the myths of “water structure” and their origins. Jacob Israelachvili has previously referred to this “structure” as a deus ex machina that can be enlisted to explain anything. However, not all such explanations need be a leap of faith. For example, the notion that water inside the cavity of the chaperonin GroEL might be non-bulk-like, because of confinement and interactions with the hydrophobic cavity walls and the GroES lid, is not obviously unlikely. Song-I Han at UCSB and colleagues explore this idea in a very nice experimental paper in which they use magnetic-resonance methods to probe the water inside the GroEL-GroES complex of E. coli (J. M. Franck et al., JACS 136, 9396; 2014 – paper here). They conclude that the density and translational dynamics of the cavity water is in fact not significantly different from the bulk. There’s a caveat that they can’t fully probe the water at the bottom of the cavity, but all the same these findings support the idea that GroEL is a “passive” cavity in which folding is much the same as it is in bulk solution.

Had I the presence of mind to have looked at Nuno Galamba’s (University of Lisbon) paper on water around hydrophobic solutes (J. Phys. Chem. B 118, 4169; 2014 – paper here) before my talk, I’d certainly have referred to it, since it supports my contention that dynamics might be more fruitful than alleged “structural” effects to understand how water is modified in such circumstances. His MD simulations suggest that the slowdown in orientational dynamics in the hydration spheres of small hydrocarbons is due primarily to the decline in hydrogen-bond acceptor switches, due to excluded-volume effects, rather than to any changes in “water structuring”, such as greater tetrahedrality.

Ariel Fernandez has advanced a very provocative claim in his continuing investigation of dehydrons, structural “defects” at protein surfaces where amide-carbonyl hydrogen bonds are imperfectly hydrated due to nanoscale confinement. These sites have a net polarization arising because the water molecules are too constrained to fully align with the electrostatic field at the protein surface. This charge is negative, says Fernandez, and may behave as a proton acceptor, i.e. it has chemical functionality. He now suggests that this basicity of dehydrons may become manifest as catalytic activity, citing the high concentration of dehydrons specifically at the active site of HIV protease (J. Chem. Phys. 140, 221102; 2014 – paper here). In other words, these structural defects turn the hydration water itself into a kind of catalytic assistant of protein function. It’s a fascinating idea, though I daresay many will want to see experimental or at least computational proof that the plausibility argument that Ariel advances actually stacks up.

Sandeep Patel, Phillip Geissler, Pavel Jungwirth and several others (forgive me for the incomplete list) have been considering how ions near the air-water interface may have specific effects on the interfacial fluctuations – a new wrinkle, perhaps, on how ions induce Hofmeister-type ion-specific effects, since such modification of fluctuations might also be expected at hydrophobic aqueous interfaces. Patel now looks more closely at this idea for the case of halide ions interacting with hydrophobin II (D. Cui et al., J. Phys. Chem. B 118, 4490; 2014 – paper here). Their simulations imply that iodide is more surface-stable than chloride – consistent with what one might expect from its greater “hydrophobicity” – and that it induces more pronounced interfacial fluctuations. In contrast, there are no significant differences in behaviour of the two ions at hydrophilic interfaces – suggesting that ion-specific effects are sensitive to the nature of the surfaces with which the ions are interacting.

More on this topic comes from Tahei Tahara and colleagues at RIKEN’s Molecular Spectroscopy Lab in Saitama (S. Nihonyanagi et al., JACS 136, 6155; 2014 – paper here). They use vibrational SFG spectroscopy to look at how counterions affect interfacial water vibrations (specifically the OH band) at charged interfaces. Here the effects seem to depend on the charge of the surfaces: at positively charged surfaces (of surfactant monolayers), the OH intensity decreases in the order of the halide Hofmeister series, whereas at negative surfaces there seems to be no such effect of the counter-cations. This seems to reflect the tendency of halides to be absorbed at the interface, whereas cation effects seem to operate via changes in the hydrogen-bond strength of the interfacial water. In other words, Hofmeister effects seem to have a different mechanism for anions and cations.

I guess there is, broadly speaking, some resonance here with a study by Yoshikata Koga at UBC in Vancouver and colleagues, who look at differences in the molecular organization of cation and anion hydration spheres (T. Morita et al., J. Phys. Chem B 118, 8744; 2014 – paper here). They use a thermodynamic methodology they have developed previously which involves addition of a cosolvent 1-propanol. They make the interesting proposal that there are five different classes of solute, which one might regard as a rather more sophisticated and physically meaningful variant of the chaotrope/kosmotrope picture. Crudely speaking, cations such as Na+ and K+ simply acquire a tight hydration shell while leaving the water beyond it unperturbed, while anions have a stronger influence with some hydrophobic character. I must say that I like this idea of trying to salvage a useable qualitative classification scheme from the confusion of the chaotrope/kosmotrope view.

Several measures of hydrophobicity have been proposed for amino acid residues, but they aren’t always consistent. There seems to be an emerging view that this is because hydrophobicity and hydrophilicity are context-dependent parameters. That idea is supported by work from Sara Bonella and colleagues at Sapienza University in Rome (S. Bonella et al., J. Phys. Chem. B 118, 6604; 2014 – paper here). They assess hydrophobicity in simulations based on the orientiation of water molecules at a certain distance from the amino acid in question, and say that a single quantity is not sufficient to characterize it. Rather, they suggest a three-parameter index, the components of which emerge from the statistical analysis of water orientation in ways that seem clear enough but which I can’t easily see how to summarize. The authors say that this method seems to work for predicting which regions of membrane proteins are the transmembrane sections.

Lei Zhou and Qinglian Liu at Virginia Commonwealth University say that adding a layer of explicit water on the surface of proteins whose normal modes are being calculated to predict anisotropic B-factors in their crystallographic structures improves the agreement with experiment (J. Phys. Chem. B 118, 4069; 2014 – paper here). It’s a nice illustration of the intimate coupling of protein and solvent.

It’s possible to engineer a buried ion pair in the hydrophobic interior of a protein without significant structural reorganization of the rest of the protein. That’s the conclusion of a study by Bertrand Garcia-Moreno E. of Johns Hopkins and colleagues (A. C. Robinson et al., PNAS 111, 11685; 2014 – paper here). They have re-engineered staphylococcal nuclease (SNase) so that it incorporates an ionizable Glu-Lys pairing (2.6 Å apart) in its interior. Although the Coulomb interaction of these largely unscreened charges is appreciable, it is not enough to offset the dehydration of the buried charges. However, two water molecules are able to penetrate deeply into the core to provide some hydration, and one of these seems able to participate in a water wire to facilitate proton transport to and from the buried ion pair. As a result, the pair is accommodated well without disrupting the protein’s structure significantly. This is useful to know because such buried ion pairs participate in some important enzymatic processes, including proton transfer and electron transfer – so there is no obvious reason why this sort of catalytic capability might not be engineered artificially into proteins.

More on the mode of operation of osmolytes: Francisco Rodríguez-Ropero and Nico van der Vegt at the TU Darmstadt say, on the basis of MD simulations, that urea stabilizes the folded state of PNiPAM via direct interactions (J. Phys. Chem. B 118, 7327; 2014 – paper here). The urea molecules enter the first hydration shell thanks to vdW interactions with the hydrophobic isopropyl groups of the polymer, creating an entropic driving force for folding via the formation of this “urea cloud”.

Irisbel Guzman and Martin Gruebele at Illinois offer a nice review of methods (especially fast relaxation imaging) for probing protein folding in vivo, where interactions with other proteins, aggregation and macromolecular crowding effects can be important (J. Phys. Chem. B 118, 8459; 2014 – paper here).

And Fabio Sterpone at the Université Paris Diderot and colleagues provide a nice review of the coarse-grained OPEP protein model for investigating all manner of cell phenomena ranging from DNA complexation and amyloid formation to crowding and hydrodynamics – the latter applied, for example, to protein unfolding (F. Sterpone et al., Chem. Soc. Rev. 43, 4871; 2014 – paper here).

Sambhu Datta at the Indian Institute of Technology and coworkers propose a comprehensive quantum-chemical treatment of the solubility of CO2 in water that includes a consideration of how hydrogen-bonding changes alter phonon energies in the fluid (T. Sadhukhan et al., J. Phys. Chem. B 118, 8782; 2014 – paper here). They say may explain how it is that RuBP in chloroplasts seems able to significantly enhance the gas solubility, increasing the rate of photosynthesis.

For anyone who wants to check out the full details (and can read French), Guillaume Jeanmairet has made available (http://arxiv.org/abs/1408.7008) his PhD thesis on a computationally inexpensive DFT treatment of water (see G. Jeanmairet et al., J. Phys. Chem. Lett. 4, 619; 2013).

Monday, June 23, 2014

Catching up in the Ruhr

The direct-interaction picture of the action of denaturants receives some support from a study by Santosh Kumar Jha and Susan Marqusee of UC Berkeley (PNAS 111, 4856; 2014 – paper here). They look at the denaturing activity of guanidinium chloride on RNase H using FRET, UV CD and kinetic measurements. They find that the initial stage of unfolding involves a fast transition to a dry molten globule, showing that it entails denaturant interactions that do not affect the solvent-accessible surface area or disrupt the hydrophobic core. It is hard to square this with any idea that GdmCl acts, at least initially, via some kind of destabilization of hydrophobic interactions.

Benjamin Schuler at the University of Zurich and coworkers also use FRET to study solvation effects on conformational changes, in this case looking at the temperature-dependent collapse of five intrinsically disordered proteins (R. Wuttke et al., PNAS 111, 5213; 2014 – paper here). These proteins become more compact as the temperature is raised. Naively this might argue for a hydrophobic, entropically driven mechanism of collapse, but the researchers argue that it’s not that simple, not least because the effect is strongest for the most hydrophilic IDP. They say that this implies a dominant contribution from temperature-dependent solvation changes for charged and polar residues, although it seems the details of that phenomenon remain to be elucidated.

There are, broadly speaking, two kinds of IDPs. One type (group a) is completely disordered, the other (group b) has regions of defined secondary structure connected by disordered stretches. Nidhi Rawat and Parbati Biswas of the University of Delhi have compared the dynamics of intermolecular and intramolecular hydrogen bonds for the two cases using MD (J. Phys. Chem. B 118, 3018; 2014 – paper here). They find that both exhibit rather similar dynamics, in both cases with the intramolecular H-bonds rather longer-lived than the intermolecular ones. But the former are somewhat more persistent in group b IDPs – perhaps as one would expect from their higher degree of structure.

Terahertz spectroscopy as a means of understanding picosecond and nanometre-scale hydration dynamics have been pioneered by Martina Havenith and her coworkers at Bochum (from where I am at this very moment returning, if the extraordinary summer storms permit…). But it has been challenging to correlate particular spectral features in this frequency range with particular molecular-scale motions. By combining THz measurements with ab initio MD simulations conducted by Dominik Marx, the Bochum group has now been able to make this connection for the case of glycine, using heavy water to identify distinct intramolecular and intermolecular vibrations, rotations and translations involving interfacial water (J. Sun et al., JACS 136, 5031; 2014 – paper here).


Proof that the RESOLV summer school in Bochum was electrifying...

It was a pleasure to meet Roland Winter again on this trip. His group at Dortmund, with collaborators in Maryland, have used neutron spin echo spectroscopy to study solvent effects on the formation of amyloid fibrils by (bovine) insulin, which happens in the presence of sodium chloride (M. Erlkamp et al., J. Phys. Chem. B 118, 3310; 2014 – paper here). They find that solvent conditions (pH, salt concentration) that promote aggregation support self-diffusion of insulin, which suggests the absence of strong concentration gradients, whereas when fibril formation is suppressed, diffusion displays the collective character diagnostic of strong concentration fluctuations. Does this seem counterintuitive to you? It does to me. But I think I see the point that stronger fluctuations imply a ‘softer’, low-compressibility system in which intermolecular interactions are rather weak.

Ariel Fernández has previously argued (J. Chem. Phys. 139, 085101; 2013) that the normal picture of the electrostatics of hydration first developed by Debye, in which water dipoles tend to align themselves with the electrostatic field, can break down at protein surfaces when sub-nanometre curvature and/or chemical heterogeneity produces constraints and frustration, leading to defects in the matrix of water-water interactions. These in turn introduces an anomalous polarization orthogonal to the electric field. During protein folding, water molecules that introduce these anomalies are driven away from the interface to minimize electrostatic energy, leaving water-exposed hydrogen-bonding groups on the protein backbone called dehydrons. In a preprint, Fernández now argues that this energy minimization drives the folding process, and that it leads to the healing of packing defects as the protein folds. The torque exerted by the protein’s electrostatic field on the water molecules at these defects inhibits water reorientation. Fernández suggests that antifreeze proteins have a particularly high density of such defects, and the resulting hindrance of water reorientation prevents the nucleation of ice at these sites.

More active water networks: Kakali Sen and Walter Thiel at the MPI für Kohlenforschung at Mülheim find using MD and quantum chemical simulations that the mechanism of the P450 enzyme CPY107A1, which catalyses a hydroxylation, involves two water networks at the active site (J. Phys. Chem. B 118, 2810; 2014 – paper here). At least one of them, based around residue E360, is involved in proton transfer to enable activation of molecular oxygen at the Fe(II) reactive site.

And again: a water wire supports proton translocation in Complex I, in which this proton pumping is redox-driven by being coupled to the movement of electrons from NADH to quinones: the first step in the mitochondrial and bacterial respiratory process. That’s the conclusion of a simulation study by Gerhard Hummer at NIH and colleagues (V. R. I. Kaila et al., PNAS 111, 6988; 2014 – paper here). The results are based on the crystal structure of Complex I from E. coli, and they imply that the water channel is formed by the cooperative hydration of three antiporter-like subunits within the membrane domain of the complex. The researchers argue that their results “suggest that water-gated transitions may provide a general mechanism for proton-pumping in biological energy conversion enzymes”, such as bacteriorhodopsin and cytochrome c oxidase.

OK, so we know that hydrated proteins can undergo a glass-like transition at low temperatures. But can essentially dry proteins do the same thing? That question is explored by Anna Frontzek at the A. F. Ioffe Physical Technical Institute of the Russian Federation in St Petersburg and colleagues (A. Frotnzek et al., J. Phys. Chem. B 118, 2796; 2014 – paper here). They look at BSA at a hydration of just 0.04 and find anomalous relaxational dynamics around 250 K, indicative of a glass-like transition even in the absence of significant hydration water.

Friday, May 9, 2014

The things internal waters get up to

I’d not previously come across guanine quadruplexes (GQs) before seeing the paper by Van Ngo and colleagues of the University of Southern California (J. Phys. Chem. B 118, 864; 2014 – paper here). These structures have been seen in human telomeres, where they can form from single-stranded DNA, but their biological role is still unclear. Telomeric GQs are stabilized by monovalent cations such as sodium, potassium and ammonium, and they have been shown capable of conducting such ions along their axis, suggesting that they can be exploited as artificial ion(-selective) channels. Ngo and colleagues investigate this process of ion conduction using MD. They find that the central channel of a GQ can host a single-file chain of water molecules, and that the passage of ions along this channel is accompanied by water molecules being “knocked out” of the chain and escaping the channel. It seems that sodium ions can move along the axis at scarcely any energetic cost, whereas for potassium there is a barrier of about 4 kcal/mol for progression from one binding site to the next. Potassium is, however, the optimal fit inside the GQ core, and so these ions are more likely to be selectively bound, while sodiums are more readily conducted.

The ion channel and water chain


How sodium ions move down the chain

Some time ago, Bertil Halle and Johan Qvist reported NMR results showing that the effect of temperature on water dynamics in the hydration shell of a hydrophobic small solute is non-monotonic (JACS 130, 10345; 2008). A molecular-scale explanation for this has never really been developed. That’s the objective of a paper by Damien Laage at the ENS in Paris and colleagues (E. Duboué-Dijon et al., J. Phys. Chem. B 118, 1574; 2014 - paper here). They have previously accounted for the retarded water dynamics in the hydration shell on the basis of excluded-volume effects that hinder local rearrangements of the hydrogen-bond network. This is the starting point for the new work (in which the solute is TMAO), but it alone is not sufficient to explain the temperature dependence. Rather, the researchers need to include an additional perturbation that describes the difference between structural fluctuations in the shell and in the bulk: at low temperatures, the constraints created by the interface with the solute impose a lower degree of structuring in the shell than is possible in the bulk.

Damien has followed this with a subsequent paper with Aoife (that’s “Eefa” to you non-Celts) Fogarty, in which they probe the reorientational dynamics of individual waters in the hydration shells of four different proteins: acetylcholinesterase, subtilisin Carlsberg, lysozyme and ubiquitin (J. Phys. Chem. B jp409805p – paper here). Despite their many differences, all of these proteins have rather smiliar hydration-shell dynamics, which the authors suggest is an indication of how the dynamics are determined by rather general features of surface chemistry and topology, which induced excluded volume effects and hinder the approach of new hydrogen-bond acceptors within the hydration network. Fluctuations of the protein surface provide an additional source of dynamic heterogeneity. The authors also explore the effects of water confinement, for example within clefts and cavities of partially hydrated subtilisin.

The water reorientational times mapped onto the surfaces of the respective proteins.

More roles for internal water molecules at ligand binding sites: Dario Estrin of the University of Buenos Aires and colleagues find that a water molecule close to the heme group of a group of mutated forms of the thermostable hemoglobin can influence the energy barriers for ligand entry and exit through steric hindrance (J. P. Bustamante et al., J. Phys. Chem. B 118, 1234; 2014 – paper here).

It seems that internal waters may also be critical for the functioning of ion channels. Michael Green and colleagues at CUNY report quantum calculations on the KV1.2 potassium channel which suggest that water molecules hydrating the ion in the channel, but not visible in X-ray structures, have a determining influence on the path of the ion (A. M. Kariev et al., Biophys. J. 106, 548; 2014 – paper here). Without an internal water network (for example, in a mutant form of the channel), the ion can get ‘stuck’, and the conductivity is much reduced. The authors also argue that protonation of the His418 residue by water is essential for gating, which might explain the observation that deuterated water slows down the gating.

How does water govern the conformations adopted by unfolded proteins? That question has gained urgency thanks to the discovery of intrinsically disordered proteins. One fact seems clear: the unfolded conformations are not arbitrary or ergodic. Reinhard Schweitzer-Stenner and colleagues at Drexel University have conducted circular dichroism and NMR studies of small peptides to get an insight into the thermodynamic factors involved, and particularly into the role of enthalpy-entropy compensation (S. E. Toal et al., J. Phys. Chem. B 118, 1309; 2014 – paper here). They find that this compensation is virtually perfect, in that there is a linear relationship between the ΔH and ΔS of solvation for glycine-terminated poly-proline, with the two cancelling exactly (ΔG=0) at about 295 K. In other words, while solvent reorganzation contributes to both the enthalpy and entropy of solvation in this (near-physiological) temperature range, it doesn’t really affect the conformational equilibria at all.

What is denaturation, anyway? Is it the same thing, regardless of how it occurs? Not according to Bruce Berne at Columbia and colleagues, who report simulations of ubiquitin which show that chemically-induced and force-induced (pulling) denaturation produce quite different states (G. Stirnemann et al., PNAS 111, 3413; 2014 – paper here). Denaturation promoted by urea produces a partly extended state with many non-native contacts, while force-unfolding creates a fully extended state with no contacts. That is perhaps what one might expect, but the full details of the conformational differences, such as differences in the dihedral angles, revealed here are not at all obvious.

Understanding the fibrous structure of cellulose in plant cell walls might be crucial to the efficient conversion of biomass to biofuels. Goundla Srinivas at Oak Ridge and colleagues say that an explicit solvent model might be needed to adequately model the transition between the crystalline and amorphous states of cellulose, and they present a coarse-grained approach for doing this (G. Srinivas et al., J. Phys. Chem. B 118, 3026; 2014 – paper here).

I have not previously come across the rhodopsin called channelrhodopsin, which differs from the light-driven ion-pump rhodopsins in that it enables passive cation conductance. How it does so is the subject of a paper by Hideki Kandori of Nagoya Institute of Technology and coworkers (S. Ito et al., JACS 136, 3475; 2014 – paper here). They say that, as with pumps such as bacteriorhodopsin, the conductance depends on a network of water molecules around the chromophore. Their FTIR spectra reveal that nine distinct water vibrational bands are implicated in the passage of cations, which they rationalize in terms of a network incorporating four bound waters around the Schiff base (shown below).


But it seems it’s not always so easy to figure out what internal water networks are up to. There is a water-filled pore threading through enzymes called family 48 cellulases, which exist primarily in bacteria and catalyse the hydrolysis of cellulose. Evidently that process consumes water, and it’s been thought that the pore might provide a channel for replenishing water at the active site. But this may not be the case, according to John Brady at Cornell and coworkers (M. Chen et al., J. Phys. Chem. B 118, 2306; 2014 – paper here). They have studied one of these enzymes, Cel48A from Thermobifida fusca, with both simulations and experimental site-directed mutagenesis, in particular looking at the effect of inserting hydrophobic groups into the pore region to disrupt the water channel. When this is done experimentally, the mutants don’t fold properly. And in the simulations, while hydrophobic residues can prevent water from filling the pore, it could find other ways of diffusing to the active site. So whether or not the water channel is functional remains unresolved.


Side view (left) and top view (middle) of the water pore structure in Cel48A, illustrating the division of the pore into rings 1, 2, 3, 4, and 5, coloured blue, red, green, orange, and yellow. On the right, only the five rings are illustrated as coloured van der Waals surfaces, along with the substrate chain.

How to quantify the hydrophobicity of protein residues is a long-standing question, and it is now clear that this depends on the structural context in which the residue finds itself. Amish Patel at U. Penn. and Shekhar Garde at Rensselaer Polytechnic Institute report a method for assigning a value of hydrophobicity that takes account of this context, based on the hydration free energy for cavity formation around the residue in question (J. Phys. Chem. B 118, 1564; 2014 – paper here). They report its use for the protein hydrophobin II, for which they provide a “hydrophobicity map” (here using a benzene-shaped “probe”, which explains the hexagonality of these images). The shape of the probe matters – a map using a small spherical probe looks slightly different from these ones.

Hydrophobicity map for hydrophobin II, from two directions

A different approach to the same end is described by Jürgen Hubbuch at the Karlsruhe Institute of Technology and coworkers (S. Amrhein et al., J. Phys. Chem. B 118, 1707; 2014 – paper here). They too attempt to develop a hydrophobicity scale that takes account of the residue’s position on the protein surface, and compare it with experiments using UHPLC. Their approach also uses (presumably hydrophobic) probe or ‘tracer’ molecules that are sensitive to the topological constraints around a given residue, quantified by a modified radial distribution function.

Water self-diffusion in salt solutions is anomalous with respect to the pure bulk (e.g. J. S. Kim et al., J. Phys. Chem. B 116, 12007; 2012). Yun Ding at ETH and colleagues show that this can be explained using ab initio MD simulations, and that it does not need any notion of “structure-making/breaking” (PNAS 111, 3310; 2014 – paper here). The ions “do not disrupt the [water] network in any significant manner”, they say. Rather, the molecular explanation is subtle, involving dynamic and electronic heterogeneity of the water molecules on diffusional timescales. I can’t help thinking that there is a moral here about the sometimes dangerous allure of physically intuitive explanations – the fact seems to be that on occasion a simple picture is merely simplistic.

The ab initio study of the geometry of the hydrogen-bond network in liquid water by Thomas Kühne and Rustam Khalliullin at the University of Mainz, on which I reported some while back, has now been extended by these authors with a comparison to the network geometry in hexagonal ice (JACS 136, 3395; 2014 – paper here). The results essentially support the previous picture, namely that “the traditional description of liquid water is fundamentally correct [but] for a significant fraction of molecules the hydrogen-bonding environments are highly asymmetric with extremely weak and distorted bonds”.

Wednesday, March 12, 2014

Weird antifreeze


The oddest finding I’ve seen recently has to be the crystal structure of the fish antifreeze protein Maxi reported by Peter Davies of the Queen’s University in Kingston, Canada, and colleagues (T. Sun et al., Science 343, 795; 2014 – paper here). This is a four-helix bundle with an interior, mostly hydrophobic channel filled with more than 400 water molecules, crystallographically ordered into a clathrate-like network of mostly five-membered rings. It seems that this ordered network extends outward through the gaps between the helices, helping to create an ordered later of water molecules on the outer surface that enables Maxi to bind to ice crystals and hinder their growth. Commenting on this work, Gerhard Hummer has called the water network a kind of molecular Velcro that holds the coils together. I have described this work in more detail in a news story for Chemistry World.

Water molecules buried deep within a protein’s interior can have extremely slow dynamics. That fact acquires functional significance in potassium channels, according to Marc Baldus of Utrecht University and coworkers (M. Weingarth et al., JACS 136, 2000; 2014 – paper here). These channels have remarkably slow recovery rates from the non-conductive to the conductive form, especially given that the macromolecular rearrangements involved don’t appear to be large. Using NMR and MD simulations, Baldus et al. find that there are several buried, ordered waters with long residence times behind the selectivity filter region of the channel, and that the recovery pathway involves exchange of these with bulk water.

Warren Beck and colleagues at Michigan State have used guanidinium as a probe of the coupling of a protein – here zinc-substituted cytochrome c – to its hydration shell (J. Tripathy et al., J. Phys. Chem. B 117, 14589; 2013 – paper here). They attribute the fluorescence Stokes shift response in the presence of Gdm ions to the enhanced flexibility of the protein-solvent network caused by direct binding of Gdm+ to the protein surface.

A far more idealized case of osmolyte effects on hydration is reported by Jens Smiatek of the University of Stuttgart, who considers how the hydration of charged model spheres is altered by urea and hydroxyectoine (J. Phys. Chem. B 118, 771; 2014 – paper here). The agenda here is the molecular mechanisms of so-called chaotropic and kosmotropic influences of osmolytes – whether, for example, these involve direct solute-cosolute or indirect (‘structure-making/breaking’) effects. It’s hard to generalize, however, about the results, other than perhaps to say that indirect effects seem to be minor and that the direct interactions of the cosolutes depends on the nature (here charge) of the solute surface. Smiatek concludes that the interactions are more complex than has often been assumed, and that “a general theory for kosmotropic and chaotropic behavior is far from being fully understood… [o]ne reason is the observed specific dependence on the considered solute surface characteristics.”

Similar issues are also explored by Abani Bhuyan and coworkers at the University of Hyderabad (P. Sashi et al., J. Phys. Chem. B 118, 717; 2014 – paper here). They use methanol titration to look at cosolvent effects on the alcohol-induced unfolding of cytochrome c at different pH, and thus differing degrees of side-chain ionization. They find that, with increasing protein charge, increasing amounts of water molecules are associated with the peptide chain, presumably because charge repulsion causes expansion of the folded state. Correspondingly larger amounts of hydration water are thus excluded by the methanol as the unfolding proceeds.

Specific ion (Hofmeister) effects on the diffusion of water at the hydration surface of a lipid bilayer are reported by Songi Han and colleagues at UCSB (J. Song et al., JACS 136, 2642; 2014 – paper here). They use Overhauser nuclear dynamic polarization to monitor water diffusion in the 2-3 layers close to the surface of a lipid vesicle, and find that various ions can have an accelerating or retarding effect that is in line with the Hofmeister series. They put the case nicely: “The concept of ions generally altering the bulk water structure, in the absence of molecular surfaces, does not seem plausible in explaining the effects of ions at the molecular level on surfaces in electrolyte solutions. However, it has been discussed in the literature that the ion’s effect on the local hydration water structure directly surrounding the ions can differ depending on the ion type”. That’s the case they make, and moreover propose a general mechanism: “This suggests that the origin of the Hofmeister ions may be the balancing between macromolecule−water and macromolecule− macromolecule interaction through the modulation of the effective surface hydrophilicity and hydrophobicity mediated by specific ions in dilute solution.”

Why, though, are water molecules generally retarded at lipid membrane surfaces in the first place? It has been suggested that the water molecules might form bridges between the lipid head groups that stabilize the membrane. This ideas is explored by Eiji Yamamoto and colleagues at Keio University in a preprint http://www.arxiv.org/abs/1401.7776. Their MD simulations indicate that water undergoes subdiffusion at a membrane surface due to binding and unbinding of the molecules in bridging conformations. The authors point out that these retarded dynamics of water might be biologically efficacious in increasing the efficiency of biomolecular binding reactions at the membrane.

In a water monolayer confined between two parallel graphene sheets, ions can induce the formation of long fluctuating chains of hydrogen-bonded molecules that can extend for up to 30 or so molecules, according to simulations by Petr Král and colleagues at the University of Illinois at Chicago (I. Strauss et al., JACS 136, 1170; 2014 – paper here). These chains can bridge two ions of opposite charge, and remain locked in place even at room temperature.

Water passing through carbon nanotubes has been found previously to have a high, almost frictionless flow rate and collective dynamics. Thomas Sisan and Seth Lichter at Northwestern now argue from MD simulations that, when the nanotubes are particularly narrow, this flow can occur in the form of solitons (Phys. Rev. Lett. 112, 044501; 2014 – paper here). The solitons are composed of defects in the single-file water chain that convect mass.