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 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.

Thursday, January 23, 2014

Does bulk water get crowded out of cells?

Although terahertz spectroscopy has become an important tool for studying biomolecular hydration, its interpretation is not straightforward. To resolve some of the ambiguities, Robert Donnan and colleagues at Queen Mary College in London have used MD to compute the vibrational density of states for several hydrated proteins of varying size, looking in particular at the distance and timescales probed by THz (O. Suchko et al., J. Phys. Chem. B 117, 16486; 2013 – paper here). They find that for all the cases studied – lysozyme, BPTI, TRP tail and TRP-cage – the hydration layer is 10 Å thick, and displays similar dynamics. Differences in the solvation dynamics for these systems seemed to stem primarily from highly retarded water molecules in the proteins’ interiors.

What dominates the solvation free energies of peptides? One view is that it is the free energy needed to create a cavity in the solvent. But this may be offset to at least some degree by the electrostatic interactions of polar groups with the water, and/or by the van der Waals interactions. To examine this balance, Montgomery Pettitt at the University of Texas at Galveston and colleagues have performed MD calculations for flexible alanine oligomers (H. Kokubo et al., J. Phys. Chem. B 117, 16428; 2013 – paper here). They find that, for rigid peptides the free-energy gains from vdW interactions more than compensate for the cost of cavity formation as the oligomers get longer. But when the solutes are flexible and allowed to collapse, this situation reverses – implying that van der Waals interactions provide a significant driving force for the collapse. It seems not yet clear, however, what role intramolecular interactions play in the collapse, since the fluctuations in that component of the free energy are large.

The role of water in the complexation of DNA-binding agents is examined for the case of the minor-groove binder netropsin by Edwin Lewis of Mississippi State University and colleagues (J. P. Ramos et al., J. Phys. Chem. B 117, 15958; 2013 – paper here). Specifically, they use calorimetry to look at how binding is affected by osmolytes (TEG and betaine), which introduce an osmotic pressure on the hydration water. The results support the earlier idea that there are two distinct binding modes of netropsin, and allow quantification of the water molecules that seemingly hydrate the bound molecule: 31 and 19 molecules for the two cases. Moreover, in the latter case at least one water molecule seems to remain trapped at the binding site, mediating the interaction with netropsin. The addition of the osmolytes, which exert broadly similar effects, has much the same effect on complexation as a reduction in the temperature.

At high concentrations, protein solutions have been found to exhibit liquid-liquid phase separation into solutions of different protein concentration. Such highly concentrated solutions are relevant to some medical conditions, such as sickle-cell anaemia and Alzheimer’s, and also to technological processes such as protein purification and storage. Johannes Möller of the TU Dortmund and colleagues relate this phase behaviour to that in protein solutions at high pressure (J. Möller et al., Phys. Rev. Lett. 112, 028101; 2014 – paper here). Using SAXS from lysozyme solutions, they find that the liquid-liquid phase separation is in fact re-entrant at high pressure. They attribute this behaviour to the effect of pressure on solvent-mediated protein-protein interactions, and conclude that pressure might be used as a means of controlling protein aggregation and crystallization.

Another possible influence on protein dynamics and function at high concentrations is crowding. Kevin Kubarych and his collaborators at the University of Michigan have been studying this matter for some time, and their latest paper (J. T. King et al., JACS 138, 188; 2014 – paper here) reports the interesting observation of a dynamical transition above a certain crowding threshold. Above this limit for lysozyme (induced by the polymeric crowding agent PEG-400, or by protein self-crowding), ultrafast 2D-IR spectroscopy reveals a significant slowdown in hydration dynamics on picosecond timescales. The authors suggest that this is a kind of jamming transition between hydration shells of the protein molecules extending out to 15-20 Å, i.e. to separations of 3-4 nm, which are certainly of the order of those found between macromolecules in cells. In other words, the transition reflects a collective frustration of rearrangements of the overlapping hydration shells. According to this picture, one would anticipate most regions of a cell to be in the “over-crowded” regime, with little “bulk-like” water. The same abrupt dynamical transition was not seen, however, in the authors’ previous studies using the small-molecule crowding agent glycerol, for which the dynamical slowdown was more gradual.

Models of the air-water interface – including models of hydrophobic hydration that invoke an interface of that nature adjacent to the hydrophobic surface – can’t in general easily accommodate a good description of long-wavelength density fluctuations, according to an analysis by Suriyanarayanan Vaikuntanathan and Phillip Geissler at the Lawrence Berkeley National Laboratory (Phys. Rev. Lett. 112, 020603; 2014 – paper here). They show that discretizing the interface, as in a lattice model, effectively suppresses long-range fluctuations, even to the extent of suppressing a roughening transition, above some critical value of the interaction potential. The authors show how one can accommodate the resulting nonlinearities, which for example allows them to describe the shape dependence of interfacial free energies.

Thomas DeCoursey of Rush University and Jonathan Hosler of the University of Mississippi Medical Center offer an intriguing discussion of the current understanding of (as the authors somewhat provocatively put it, the “philosophy of”) voltage-gated proton channels (J. R. Soc. Interface 11, 20130799; 2014 – paper here). The paper includes an overview of such issues as proton hopping along water wires, and mechanisms for proton selectivity (for example, exclusion of alkali metal ions) and for the suppression of proton transport in aquaporins.

Christopher Fennell at Oklahoma State University and colleagues have previously developed a computationally inexpensive water model called the semi-explicit assembly (SEA) model, which does a good job of calculating the solvation free energies of polar and nonpolar solvents (Fennell et al., PNAS 108, 3234; 2011). They now extend the SEA model to a version they call field-SEA, which can handle ions and charged solutes with no additional computational overhead (L. Li et al., J. Phys. Chem. B jp4115139; 2014 – paper here).

Thursday, December 12, 2013

Enthalpy/entropy compensation

The weak forces that govern protein-protein association and aggregation are of course intimately connected with hydration, partly for example via hydrophobic interactions but also through the electrostatic consequences of water removal at the interfaces. These effects are examined in a continuum solvent model by Sergio Hassan at the NIH in Bethesda and colleagues (A, Cardone et al., J. Phys. Chem. B 117, 12360; 2013 – paper here). They use the model to examine the barnase-barstar complex, and say that it could be extended to multi-protein systems.

A rather exquisite water-mediated mechanism of enzymatic activity is reported by Qiang Cui at Wisconsin-Madison and colleagues (P. Goyal et al., PNAS 110, 18886 – paper here). They describe a computational study of cytochrome c oxidase, which uses oxygen reduction to pump protons across a membrane. During this process a glutamate residue is believed to act as a temporary proton donor. Cui and colleagues show that the proton affinity of this Glu is controlled by the degree of hydration in an internal hydrophobic cavity, which is itself governed by protonation of a substituent on the heme group 10Å away, triggering movement of a loop gating the cavity’s entrance.

Enthalpy/entropy compensation in protein-ligand binding is the topic of a nice study by George Whitesides’ group in a special issue of the European Physical Journal (doi:10.1140/epjst/e2013-01818-y – paper here). The group examines this issue further in a paper in JACS (B. Breiten et al., JACS 135, 15579; 2013 – paper here), where they look at the human carbonic anhydrase system that they have also studied earlier (see P. W. Snyder et al., PNAS 108, 17889; 2011). This new paper expands on that earlier work, in which a series of ligands modified with various thiazole-based sulfonamides was used to probe the effects of solvent rearrangements within the binding cavity. They find that the changes in the free energy of binding, and the contributions from enthalpy and entropy, are largely determined by the rearrangements or displacements of water, and voice a telling conclusion: “This water- centric view of ligand binding – and H/S-compensation – cannot be rationalized by the lock-and-key principle and suggests that the molecules of water surrounding the ligand and filling the active site of a protein are as important as the structure of the ligand and the surface of the active site.”

Myoglobin binds various ligands, such as O2, CO and NO, in an internal cavity. It also binds water in internal sites, but crystallographic studies have given conflicting views on where these cavities are and how occupied they are. Shuji Kaieda and Bertil Halle at Lund have used deuteron and 17O magnetic relaxation dispersion spectroscopy to probe these hydration sites and the dynamics of the water molecules occupying them (J. Phys. Chem. B 117, 14676; 2013 – paper here). They find that the waters come and go on a microsecond timescale in all four sites, despite their significant separation, and conclude that these dynamics share a global mechanism involving transient penetration of the protein by hydrogen-bonded chains that may intermittently ‘flush’ the cavity network.

More on small-molecule solute perturbations to protein structure: Warren Beck and colleagues at Michigan State University report fluorescence spectroscopic measurements on zinc-substituted cytochrome c in the presence of guanidinium ions, in an effort to discover why Gdm+ perturbs the protein dynamics by making it apparently more flexible (J. Tripathy et al., J. Phys. Chem. B 117, 14589; 2013 – paper here). They conclude that part of this response comes from a change in structure of the protein’s hydration shell because of direct binding of Gdm+ to the protein surface.

How do magnesium ion channels attain their high specificity in the presence of high concentrations of calcium? Todor Dudev at the Academia Sinica and Carmay Lim at National Tsing Hua University in Taiwan say that the differences in the metals’ hydration shells are the key (JACS 135, 17200; 2013 – paper here). Their calculations suggest that the hexacoordinated magnesium ions polarize the bound water more strongly, resulting in stronger ion-water-protein interactions.

How water diffuses at the surface of a lipid membrane has been studied before, but it can be hard to ensure that just the water molecules immediately at the interface are probed. Robert Bryant at the University of Virginia Charlottesville and colleagues claim to be able to do that using magnetic relaxation dispersion spectroscopy for phospholipid vesicles in deuterated water (K. G. Victor et al., J. Phys. Chem. B 117, 12475; 2013 – paper here). They report that the water molecules explore the interface via essentially two-dimensional diffusion over the vesicle surface, with a translational correlation time that seems relatively long: about 70 ps. This modified dimensionality of water motions, they say, stems from an excluded-volume effect.

The molecular basis of anaesthesia is still unclear, although it seems to involve low-affinity binding of the anaesthetic to proteins. Hai-Jing Wang of UNC and colleagues say that hydration water plays a crucial role in this process (H.-J. Wang et al., J. Phys. Chem. B 117, 12007; 2013 – paper here). They use NMR to look at the binding of volatile halogenated alkanes (known anaesthetics) to BSA, which has binding pockets for such molecules. They find that binding occurs only once the hydration level of the protein surpasses a critical threshold. The molecular details of what this hydration water does remain to be elucidated, but Wang et al. suppose that the threshold hydration level is needed to “establish a favourable free-energy landscape for the binding of anaesthetics to the pre-existing binding sites.”

Peter Rossky and Lauren Kapcha at Texas at Austin have introduced a new hydrophobicity scale for protein constituents that works at the level of individual polar and non-polar atoms rather than classifying each residue as a whole (J. Mol. Biol. 10.1016/j.jmb.2013.09.039 – paper here). They say that it gives an appropriate measure of hydrophobicity in cases where a residue-based approach fails – without being any more computationally intense – and that it shows that this atomistic level of detail is therefore sometimes indispensable.

Another computational challenge is addressed by Sergio Pantano of the Institut Pasteur de Montevideo in Uruguay and coworkers (H. C. Gonzalez et al., J. Phys. Chem. B 117, 14438; 2013 – paper here). They describe a way to combine the common SPC, TIP3P and SPC/E atomistic water models with a coarse-grained model called WatFour or WT4, demonstrating its effectiveness via (among other things) simulations of the β1 domain of streptococcal protein G.

There’s a curious and interesting paper by Akira Yamakata of the Toyota Technical Institute in Nagoya and colleagues on the observation of hydration shells of ions being destroyed by electrochemical control of the ions’ binding to an electrode surface (A. Yamakata et al., JACS 135, 15033; 2013 – paper here). The researchers use a platinum electrode rendered hydrophobic by coverage with a CO monolayer, and use time-resolved IR spectroscopy to monitor the hydration shells of tetrapropylammonium (Pr4N) and sodium ions as they are pulled onto the surface by the electric field. They can monitor the destruction of the hydration shells as this happens, and say that while that of Pr4N is rather easily destroyed at high field so that the ion interacts directly with the CO layer, the shell of sodium is more rigid and remains intact.

Lawrence Pratt and colleagues at Tulane University use simulations to demonstrate that the hydrophobic interactions between hard-sphere solutes in water are more attractive and endothermic than is predicted by the molecular-scale Pratt-Chandler theory (M. I. Chaudhari et al., PNAS 10.1073/pnas.1312458110 – paper here). This shows what an improved theory will have to shoot at.

Peter Hamm and coworkers at the University of Zurich have used 2D Raman-THz spectroscopy to probe collective intermolecular modes of pure liquid water, and conclude that there are different types of hydrogen-bonded networks – albeit only on a very short (100-fs) timescale, which is not compatible with controversial proposals for persistent heterogeneity of these networks like that of Anders Nilssen et al. (J. Savolainen et al., PNAS 10.1073/pnas.1317459110 – paper here). This seems to add to the growing view that this apparent controversy is best seen as a question of the different timescales being examined, and that at room temperature any ‘two-state’ picture is very rapidly averaged away.

Finally, a curiosity about the macroscopic mechanical roles of water in plants. Peter Fratzl of the Max Planck Institute of Colloids and Interfaces in Potsdam and colleagues look at how changes in hydration can produce stresses and movement in plants via swelling and shrinkage (L. Bertinetti et al., Phys. Rev. Lett. 111, 238001; 2013 – paper here). The free energy needed for such actuation can be considered to be provided, at least in part, by the formation of new hydrogen bonds. The authors propose a model that allows them to estimate the free energy made available by water absorption into woody plant tissue, and find that (except in the case of nearly dry tissue) it amounts to about 1.2 kT per water molecule, suggesting that the water bound to the macromolecules in the tissue acquires about one additional hydrogen bond for every eight molecules. Frankly, I don’t fully understand the arguments here: the authors say that “This would suggest that the main driving force for water absorption in non living plant tissues is a phase transition of water to a liquid state, characterized by a stronger H-bond network, occurring when H2O is confined within the macromolecular components of the wood cell wall material.” I’m going to have to get back to you on that.

Thursday, November 14, 2013

Using water for drug design

Alfonso García-Sosa at the University of Tartu in Estonia has published a paper getting to grips with precisely the question that I have long wanted to see addressed: how to employ water molecules in design strategies for drug binding (J. Chem. Inform. Model. 53, 1388; 2013 – paper here). He has analysed over 2,000 crystal structures of hydrated and non-hydrated ligand-receptor complexes (including many drugs), and finds that bridging water molecules are an effective strategy for tight binding. He concludes that such a binding mechanism could be beneficially targeted, and that “if a tightly bound, bridging water molecule is observed in the binding site, attempts to replace it [in a designed drug that binds competitively] should only be made if the subsequent ligand modification would improve also its ligand efficiency, enthalpy, specificity, and pharmacokinetic properties.” This is just the kind of large-scale study that is needed to extract the advantages or otherwise, and the effective modes, of using water molecules in ligand design.

It seems increasingly likely that the mechanism of hydrophobic association, once considered in terms of a static picture involving expulsion of ‘bound’ water, is in fact associated with dynamical effects, in particular the fluctuations in the hydrogen-bonded network around the hydrophobes. That view is emphatically supported in a paper by Aljaz Godec and Franci Merzel of the National Institute of Chemistry in Ljubljana, working with Jeremy Smith at Oak Ridge (Phys. Rev. Lett. 111, 127801; 2013 – paper here). Their MC simulations of two hydrophobic particles (of about the size of a methane molecule) coming together shows that crossing over the desolvation barrier to the associated state involves a large collective fluctuation in hydration water in which the intervening hydrogen-bonded clusters are mostly displaced from the inner to the outer hydration shell. As the authors conclude, “a complete description of hydrophobic association can be obtained only by explicitly considering collective fluctuations involving many-body correlations between water molecules.”

The observation by Amish Patel, Shekhar Garde and their colleagues that hydrophobic association is faster near a hydrophobic surface, which I have mentioned before in this blog, is fleshed out in detail by these researchers in a new paper (S. Vembanur et al., J. Phys. Chem. B 117, 10261; 2013 – paper here). The key issue is that the interfacial water, like that at the air-liquid interface, is more easily displaced, lowering the desolvation barrier to association. Naturally, this means that hydrophobic surfaces might be used to catalyse aggregation processes. I wonder if it might also help to explain Barry Sharpless’s observations some years back of an increase in organic reaction rates at the air-water interface?

Water diffusion close to lipid membranes is not like that close to solid surfaces, Roland Netz at the Free University of Berlin and colleagues say (Y. von Hansen et al., Phys. Rev. Lett. 111, 118103; 2013 – paper here). Their MD simulations reveal that, whereas at solid surfaces water diffuses faster laterally than perpendicularly, at lipid membranes the opposite is true. This seems to be because the lipids present a rough surfaces, with some head groups protruding from the leaflets, so that lateral diffusion takes place amidst a rough free-energy landscape and is correspondingly hindered. As a result, lateral motion in the fluid phase at the membrane surface involves a distinct perpendicular component to the trajectories: water molecules wander in local energy traps before moving up and away an then “descending” elsewhere.

The dynamics of interfacial water at lipid membranes is also probed by Kevin Kubarych and colleagues at the University of Michigan using IR spectroscopy (D. G. Osborne et al., J. Phys. Chem. B jp4049428 – paper here). They infer this from the spectral diffusion of a labelled cholesterol derivative in the membrane, which reflects the dynamics of its hydration water, and find that these dynamics are three times slower than those of the same molecule in bulk water. The paper largely establishes this as a nice tool that might be used to look at variations in water dynamics at different locations in a membrane.

One explanation for the apparent long-ranged hydrophobic interaction measured between adsorbed monolayers in the surface force apparatus invokes electrostatic interactions between the restructured monolayers themselves (see, for example, Jacob Klein’s work: Phys. Rev. Lett. 96, 038301; 2006 and 109, 168305; 2012). Max Berkowitz and colleagues at UNC investigate this idea using a lattice model to look at how a surfactant coated charged (mica) surface might become reconfigured into heterogeneous structures (C. Eun et al., J. Phys. Chem. B jp405979n – paper here). They find that indeed an initially uniform surfactant monolayer can become transformed into a non-uniform surface covered with patches of surfactant bilayer separated by patches of bare (water-covered) mica. This simple model system might help to understand not just Klein’s studies but also the somewhat related issue of formation of lipid rafts in mixed-lipid membranes.

Another ongoing discussion is the mechanism by which osmolytes stabilize proteins. Piotr Bruzdziak and colleagues at Gdansk University of Technology in Poland examine that issue by using IR spectroscopy and DSC to study the effects of various osmolyes (TMAO, Gly, NMG, DMG) on the stability of lysozyme (P. Bruzdziak et al., J. Phys. Chem. B 117, 11502; 2013 – paper here). In contrast to many recent simulation studies of this problem, the authors here conclude that the osmolyte-protein interaction is indirect, the osmolytes “enhancing” water structure with a consequent “tightening” of protein folding.

OK, why not touch on another controversial topic too: the molecular basis for the dormancy of bacterial spores, and in particular whether this involves an ‘altered state’ of water. That’s addressed in a preprint ( by Bertil Halle at Lund and colleagues, using deuteron magnetic relaxation dispersion to look at the state of water in spores of B. subtilis. Two models for the structure of the core aqueous phase have been proposed: a gel, in which mobile water permeates a macromolecular network, and a glass, in which everything inside the cells (including water) forms a solid amorphous phase. The NMR results clearly support the former picture – the water remains mobile, albeit with rotational motion about 15 times slower than the bulk.

Calcium release-activated calcium (CRAC) channels play an important role in cell signalling, and their dysfunction is associated with cardiac arrythmia and immunodeficiency problems. Thanks to a recent crystal structure of the pore region of a CRAC channel, Michael Klein and colleagues at Temple University have been able to investigate the mechanism of ion permeation through simulations (H. Dong et al., PNAS 110, 17332; 2013 – paper here). They say that a central hydrophobic region of the channel is the crucial region for switching on and off, and that it is the hydration of this region that controls ion transport: a small change in the number and orientation of water molecules here significantly alters the local electrostatic field. In other words, this is another channel that is water-regulated.

Moving onto another channel: the high water permeability of aquaporins may be a result of an optimization of shape at the pore entrance to minimize viscous dissipation, according to a study by Laurent Joly of the University of Lyon 1 and colleagues (S. Gravelle et al., PNAS 110, 16367; 2013 – paper here). Their finite-element calculations suggest that the hour-glass profile of the pore is particularly efficient at reducing dissipation, and that the optimal opening angle of 5-20 degrees is in the range of those observed in the proteins.

Aquaporins have of course a well resolved crystal structure, but many membrane proteins are intrinsically disordered, especially in the regions that extend beyond the lipid membrane and into the extracellular matrix. Songi Han of UCSB and colleagues suggest that hydration dynamics – in their case as measured by Overhauser dynamic nuclear polarization NMR – can provide a proxy for at least locating these extended protein segments (C.-Y. Cheng et al., PNAS 110, 16838; 2013 – paper here). The technique relies on the apparent existence of a gradient of diffusion dynamics up to 3 nm away from the membrane surface (really?). The authors can seemingly also use the technique to look at protein structure parallel to the membrane surface, as in the case of an amyloid-forming protein called alpha-synuclein associated with Parkinson’s disease. They report a sinusoidal variation in water retardation close to the lipid surface which they interpret as the result of the alpha-S coiled into an alpha helix embedded laterally in the membrane with its axis about 1-3 Å below the phosphate head groups, while the C-terminus end floats freely and disordered above the membrane surface.

Water dynamics generally found to be retarded in the vicinity of proteins. But NMR measurements on crystals of the protein Crh (protein catabolite repression Hpr) have indicated that water in the crystals may remain mobile down to at least -30 C – in other words, it seems more mobile than bulk water at these temperatures (Böckmann et al., J. Biomol. NMR 45, 319; 2009). How can this be? To answer that question, Anja Böckmann of the University of Lyon, who did the NMR wirk, has collaborated with Wilfred van Gunsteren at ETH Zurich and colleagues to conduct MD simulations of the crystalline form of Crh (D. Wang et al., J. Phys. Chem. B 117, 11433; 2013 – paper here). They find that indeed while retarded dynamics of hydration water are seen at 291 K, at 200 K (close to the glass transition) the rotational and translational mobility of this water is greater than in bulk. But while introducing artificial perturbations such as rigidifying the protein or switching off protein-solvent electrostatics does induce some enhanced mobility of either translation or rotation, the cause of the overall water mobility at this temperature remains obscure.

While on the subject of glassy low-temperature behaviour, Thomas Loerting of the University of Innsbruck and colleagues have reported a second glass transition in amorphous ice at 116 K, which they say is related to the coexistence of two highly viscous metastable liquid-like phases: a low-density phase with a glass transition of 136 K and a high-density phase which undergoes this second glass transition at 116 K (K. Amann-Winkel et al., PNAS 110, 17720; 2013 – paper here). Thus, they say, the putative HDL and LDL “can both be observed experimentally” – although the results don’t directly address the question of whether the two can be interconverted by a first-order liquid-liquid transition ending at a critical point.

A stark and rather remarkable reminder of how much still remains to be understood about the hydrogen bond is provided by Michele Ceriotti at Oxford and colleagues (PNAS 110, 15591; 2013 – paper here). Their ab initio simulations that incorporate nuclear quantum effects show that the protons in water’s hydrogen bonds undergo fluctuations that take them towards the oxygen acceptor atoms for significant fractions of time, sometimes leading to spontaneous proteolysis. What’s more, these events are strongly correlated among neighbouring hydrogen bonds, so that perturbations to the hydrogen-bonded network seem likely to modulate this distinctly non-classical effect in significant ways.

The discussion continues of how ions alter the dynamics of water in their hydration shells. MD simulations by Ana Vila Verde and Reinhard Lipowsky at the MPI for Colloids and Interfaces in Potsdam look at the effect of ion pairs (here magnesium sulphate and caesium chloride) to see to what extent the slowdown of water dynamics in cooperative between the anion and cation (J. Phys. Chem. B 117, 10556; 2013 – paper here). They find that the cooperative slowdown can be intense (especially for ions of high charge density), but only in the first hydration shell. I note their comment “our results do not support the notion that the Hofmeister series is due primarily to long-range effects of ions on water properties. Instead, they point to the possibility that models of ion solvation may focus primarily on the first hydration layer without excessive loss of accuracy.”

Directional transport of water through a carbon nanotube connecting two reservoirs, driven by electric fields, has been reported in simulations by several teams over the past few years. Hangjun Wu of Zhejiang Normal University in Jinhua and colleagues now add another instance (X. Zhou et al., J. Phys. Chem. B 117, 11681; 2013 – paper here). They show that a vibrating charge that deforms the nanotube away from its halfway point (thus introducing the asymmetry that underpins the ratchet-like effect) will create a directional flux of water. But the direction of that flux is dependent on the displacement of the nanotube wall, and can even change sign when the displacement gets very large.

Daryl Eggers at San José State University continues to develop his picture of solvation water as a reactant in solution equilibria. With Brian Castellano he considers what happens when one includes in the thermodynamic treatment of binding equilibria a representation of how water is perturbed next to solutes (so that it is not ‘equivalent’ on both sides of the equilibrium) (B. M. Castellano & D. K. Eggers, J. Phys. Chem. B 117, 8180; 2013) – paper here). The spirit of the exercise is nicely encapsulated thus: “When water is viewed as a coreactant, it becomes apparent that the traditional equation for the standard-state Gibbs free energy of binding represents an unbalanced equation.” In fact, this means that the equilibria and binding constants become concentration-dependent. Perhaps this might account for discrepancies between different ways of experimentally measuring the enthalpy changes of some reactions.

To what extent can the vibrations of liquid water be decomposed into essentially single-molecule bending and stretching OH modes, plus intermolecular contributions? Less than was thought, according to Andrei Tokmakoff, currently at the University of Chicago, and colleagues (K. Ramasesha et al., Nat. Chem. 5, 935; 2013 – paper here). Their studies using ultrafast 2D IR spectroscopy show that there is strong mixing between all of these modes, so that all the vibrations have some collective component. This will require some significant rethinking of relaxation and energy dissipation in water.

Thursday, October 10, 2013

In praise of the Nobels

All the three recipients of this year’s Chemistry Nobel Prize – Martin Karplus, Arieh Warshel and Michael Levitt – have of course played some part in the general exploration of water’s roles in molecular biology. But Levitt in particular has been inspirational. It was what he wrote with Mark Gerstein in Scientific American in 1998 that, more than anything else, persuaded me there was a story to be unfolded about the importance of water in biology:
“When scientists publish models of biological molecules in journals, they usually draw their models in bright colors and place them against a plain, black background. We now know that the background in which these molecules exist – water – is just as important as they are.”

Much of the popular discussion of the Nobel award has centred around the technical computational aspects of these guys’ work, almost as though they deserve praise as programmers. But it is comments like this one that reveal the deep chemical insight behind all that computer stuff. This why I view the decision with deep satisfaction.

Monday, October 7, 2013

Water and protein folding

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

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

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

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

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

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

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

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

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

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

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

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

Tuesday, August 20, 2013

Spectral tuning and pumping in rhodopsins

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

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

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

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

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

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

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

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

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

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

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

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