Friday, October 28, 2016

Are hydrophobic protein surfaces like big or small hydrophobes?

It seems to me that a paper on protein denaturation by Michele Vendruscolo at Cambridge, Stefano Gianni at the University of Rome La Sapienza, and their colleagues will repay careful study (C. Camilloni et al., Sci. Rep. 6, 28285; 2016 – paper here). The researchers use MD simulations to model observed NMR shifts during hot and cold denaturation, and thereby to gain insight into transition-state structures, changes in hydration and thermodynamic parameters. This enables them to characterize in some detail the differences between hot and cold denaturation: the former has more secondary structure, being more influenced by hydrophobic interactions. In effect this points to the existence of two alternative folding mechanisms from denatured states. What’s more, water molecules at the protein surface have the same number of hydrogen bonds on average as those in the bulk. This is what theory predicts for small hydrophobes (<1 nm or so), whereas for larger extended hydrophobic surfaces some hydrogen bonding is thought to be inevitably lost. In other words, it seems that the hydrophobicity of proteins must, on account of their complex surface topography and chemical heterogeneity, be considered to be more akin to that of small rather than large hydrophobes.

A detailed look at how protein surface topography and chemistry affects local water organization is described by Tom Kurtzman of Lehman College and coworkers (K. Haider et al., JPCB 120, 8743; 2016 – paper here). They look at the ligand-binding clefts of six structurally diverse proteins and identify circumstances where the constraints on local water structure compromise the enthalpy. These include deep and narrow cavities and ones with weak water-solute interactions, but also sometimes sites with charged residues in which there can be frustration of adjacent water-water interactions. Clearly this kind of information should be useful for designing ligands that bind competitively to a site, but it remains unclear whether one can yet identify generic design features or whether each case must be considered on its own terms. The broader question is perhaps whether the hydration characteristics must be considered to have been selectively fine-tuned or simply a necessary compromise occasioned by conflicting demands on the binding site. That’s to say, are the hydration environments each individually ‘functional’ or at least partly an epiphenomenon?

One of the clearest cases I can remember seeing for the coupling of hydration structure with protein motions and function is supplied by Tomotaka Oroguchi and Masayoshi Nakasako at Keio University (Sci. Rep. 6, 26302; 2016 – paper here). They have looked at the hexameric multi-domain protein glutamate dehydrogenase (GDH) using MD simulations and AFM. The opening and closing of a hydrophobic pocket HS1 are accompanied by wetting and drying of the pocket, while binding and unbinding of water molecules in a hydrophilic crevice HS2 accompany changes in its length. These two changes in hydration are coupled, creating a kind of hydration-driven mechanism for large-scale conformational change in GDH.

Drying and wetting of the hydrophobic pocket HS1 accompanying the opening and closing of the cleft.

Conformational changes in the GDH protein due to coupled hydration changes at the sites HS1 and HS2.

The coupling of hydration change to large-scale protein dynamics is also the subject of an experimental study by Keisuke Tominaga and colleagues at Kobe University using dielectric spectroscopy at THz frequencies (N. Yamamoto et al., JPCB 120, 4743; 2016 – paper here). They look at lysozyme in the solid state under different hydration conditions, and see two relaxational modes. They attribute the faster of them, with a ~20 ps relaxation time, to coupled water-protein motion: the mode is primarily due to hydration water dynamics, but the hydration water “drags” with it the hydrophilic groups at the protein surface. The slower (~100 ps) mode might be due to motions of the amino-acid side-chains induced by hydration. Looking at the temperature dependence of the spectra, the authors also see a signature of the familiar dynamical transition around 200 K.

The same issue is explored by Dongping Zhong and colleages at Ohio State University using femtosecond spectroscopy of tryptophan relaxation (Y. Qin et al., PNAS 113, 8424; 2016 – paper here). They too see coupling between hydration water and protein side-chain dynamics which slows down the water reorientational relaxation at the interface relative to the bulk. (They study DNA polymerase IV.) Mutational studies and MD simulations imply that causation here goes in the direction of the protein side-chain fluctuations being slaved to the cooperative dynamics of the hydration water.

A closer look at the dynamics of hydration water is provided by Peter Bolhuis of the University of Amsterdam and colleagues, who compare MD simulations with femtosecond IR spectroscopy of the hydration of bovine α-lactalbumin, both in its native and a misfolded state (Z. F. Brotzakis et al., JPCB 120, 4756; 2016 – paper here). The water relaxation times here are typically of the order of tenths of to several picoseconds; ‘slow’ waters have relaxation times > 7 ps, some as much as 20 ps. These waters tend to be located in concavities on the protein surface and make fewer hydrogen bonds with surrounding waters than do molecules in the bulk. Moreover, waters near hydrophobic groups tend to be slower on average than those near hydrophilic groups. But although misfolding exposes more of the hydrophobic surface, it also means that these hydrophobic regions are less concave, and so the water dynamics is somewhat faster on average and there are fewer of the “ultraslow” sites.

Water reorientational decay times seen in simulations of native (left) and misfolded (right) bovine α-lactalbumin.

A somewhat comparable exercise is conducted for B-DNA by James Hynes and Damien Laage at the ENS Paris and colleagues (E. Duboué-Dijon et al., JACS 138, 7610; 2016 – paper here). And there are some commonalities: while the hydration water is generally rather slower to reorient than in the bulk, the waters confined in the narrow minor groove are much more significantly retarded (relaxation times 30-85 ps). Moreover, there is considerable heterogeneity, and some of this comes from coupling of the macromolecular fluctuations with the water dynamics, especially in the minor groove. In other words, there does not seem in this case to be slaving of biomolecular dynamics to those of the solvent, but more or less the reverse.

Water reorientational times on the minor and major grooves of the B-DNA dodecamer (CGCCAATTCGCG)2

Lorna Dougan at Leeds and colleagues have found evidence of a low-density form of water at low temperatures (285-238K), which might be related to the putative phase transition separating low- and high-density liquids in the metastable regime (J. J. Towey et al., JPCB 120, 4439; 2016 – paper here). They keep the water liquid by mixing it with the cryoprotectant glycerol. Neutron scattering and simulation show that at low temperatures the mixture segregates at the nanoscale, and the water nanophase has greater tetrahedral ordering than the bulk.

Predicting protein structure from sequence data often draws on information on homologous structures or fragments from the Protein Data Base. But such homologies cannot always be spotted, or might not be present in the database, or might not be reliable. Peter Wolynes and colleagues at Rice have developed a scheme for predicting structures ab initio, without bioinformatics input, using what they call the atomistic, associative memory, water mediated structure and energy model (AAWSEM) (M. Chen et al., JPCB 120, 8557; 2016 – paper here). This uses coarse-grained simulations at the whole-protein level while drawing on atomistic simulation of fragments – and crucially, incorporates water-mediated interactions in the folding process. It’s a smart approach to the folding problem that draws on the biological reality – the fact that protein folding is funneled to make it evolutionarily robust to small variations in sequence – rather than brute-force number-crunching.

Water mediation is thought to be important too for the aggregation of amyloid fibrils. Samrat Mukhopadhyay and colleagues at the Indian Institute of Science Education and Research in Mohali have used time-resolved fluorescence measurements on the human prion protein (PrP) to investigate how (V. Dalal et al., ChemPhysChem 17, 2804; 2016 – paper here). They find that water hydrating the amyloid-competent oligomers has mobility retarded by three orders of magnitude relative to the bulk, perhaps because of entrapment in the collapsed polypeptide chains. They say that this water might create a hydrogen-bonded network that stabilizes the partly unfolded, molten oligomer conformation and acts as a scaffolding for the assembly of oligomers into fibrils.

Proposed role of ordered water molecules in the misfolding and amyloid formation of PrP – and in protein misfolding diseases more generally.

Antiviral drugs against influenza B could work by blocking the proton-conducting channel BM2, but no such have yet been devised. Mei Hong at MIT and colleagues have used NMR to investigate the mechanism of proton transport in BM2 and the role of hydration, and to elucidate the differences with AM2 from influenza A (J. K. Williams et al., JACS 138, 8143; 2016 – paper here). The His19 residue in BM2 remains unprotonated to lower pH than the corresponding His 37 in AM2, but increasing channel hydration in acidic conditions seems to enhance proton transport to His 19 from water molecules.

Why trehalose acts as a cryoprotectant of protein structure still isn’t fully understood. Jan Swenson and coworkers at Chalmers University of Technology in Göteborg try to develop a comprehensive picture by looking at how trehalose affects the protein glass transition, denaturation temperature, and solution viscosity (C. Olsson et al., JPCB 120, 4723; 2016 – paper here). They study the myoglobin-trehalose-water system using DSC and viscometry. In short, their results seem to exclude the picture in which trehalose displaces water in the solvation shell; on the contrary, they suggest that the protein retains one or two layers of water within a stabilizing water-trehalose matrix. This would be consistent with an apparent lack of coupling between the trehalose-water matrix dynamics and the stability of the native protein.

Schematic of the interactions between water, trehalose and protein.

That picture of a lack of direct interaction between trehalose and proteins – the disaccharide is in fact preferentially excluded from the protein hydration layer – is also the general context for an experimental study by Christina Othon of Wesleyan University in Connecticut and colleagues of trehalose bioprotection (N. Shukla et al., JPCB 120, 9477; 2016 – paper here). Using ultrafast fluorescence spectroscopy for two fluorescent probes, they see a slowdown of water reorganizational dynamics at relatively low trehalose concentrations (0.1-0.25 M, well below the vitrification threshold). At these concentrations, there is around 7 water layers between osmolyte molecules. These results therefore support an indirect mechanism for cryoprotection. Sucrose has much the same effect, but less markedly, the researchers say.

The interaction between two hydrophobic particles in water is generally attractive: this is simply the (water-mediated) hydrophobic effect. But Alenka Luzar and coworkers at Virginia Commonwealth University show that this interaction can become repulsive (B. S. Jabes et al., JPC Lett 7, 3158; 2016 – paper here). Such repulsion has been seen before in simulations of fullerenes and carbon nanotubes in water, and has sometimes been attributed to specific structural changes in the water. But Alenka and her colleagues show that it can be explained purely as a geometric effect of the thermodynamic cost of formation of a liquid-vacuum interface bridging the hydrophobic particles (in these calculations, pure and propyl-terminated graphitic nanoparticles) when drying occurs in the intervening space. This process can be modeled with a straightforward, bulk-like Young-type calculation of the surface free energies.

Nanoconfinement effects on water structure and properties are investigated by Vrushali Hande and Suman Chakrabarty of the National Chemical Laboratory in Pune through simulations of water inside reverse micelles and water-in-oil nanodroplets (Phys. Chem. Chem. Phys. 18, 21767; 2016 – paper here). For the reverse micelles the interface is (negatively) charged, and the deviations from bulk-like behaviour are longer-ranged for orientational order than they are for translational ordering. These effects are far less pronounced for nanodroplets in oil, where the interface is hydrophobic, indicating that electrostatic influences on the hydrogen bonding are more pronounced than spatial confinement per se.

Also on nanoconfinement: quite why water has an enhanced mobility in carbon nanotubes remains a matter of some debate. Using IR spectroscopy, Pascale Roy at the Synchrotron Soleil in Gif-sur-Yvette and colleagues suggest that it may be due to unusually “loose” hydrogen-bond networks among water molecules inside the nanotubes (S. D. Bernadina et al., JACS 138, 10437; 2016 – paper here). They look at nanotubes with diameters of 0.7-2.1 nm, in which the water varies from single-file chains to multilayers, and find a spectroscopic signature of “loosely bonded water” in all cases – in the latter seeming to correspond to waters in the outer layers with dangling OH bonds pointing towards the nanotube walls.

The distance dependence of the hydrophobic force between two hydrophobic walls is investigated in MD simulations by Biman Bagchi and colleagues at the Indian Institute of Science in Bangalore (preprint arxiv.1608.04107). They find a bi-exponential force law, with correlation lengths of 2 nm and 0.5 nm, and a crossover close to 1.5 nm. This behaviour is mimicked by the tetrahedral order parameter, but I’m not entirely clear what the authors’ mechanistic explanation is.

Of course, the issue with many studies of this kind is that your results might only be as good as your model. Angelos Michaelidies and colleagues at UCL offer an overview of the extent to which density-functional theory supplies a good description of water, from small clusters to the bulk (M. J. Gillan et al., JCP 144, 130901; 2016 – paper here). In particular they consider how well different functional forms of exchange-correlation terms perform, and what role many-body terms play. Looks like essential reading for anyone using DFT to model aqueous systems.

Many-body effects are also central to a study by Shelby Straight and Francesco Paesani at UCSD of influences of water’s dipole moment on the hydrogen-bond network of pure water (JPCB 120, 8539; 2016 – paper here). They use simulations to predict the infrared spectra of HOD in H2O, and in particular the shape of the OD stretch. They find that the calculated spectral diffusion of this vibrational frequency depends rather strongly on exactly how one truncates a many-body expansion of the water dipole.

How effectively can hydration be described with a coarse-grained model? Bill Jorgensen and colleagues consider the performance of one attempt to balance accuracy and speed that mixes all-atom and coarse-grained descriptions – the so-called AAX-CGS model, in which all-atom solutes are solvated with coarse-grained water (X. C. Yan et al., JPCB 120, 8102; 2016 – paper here). The approach works well for hydrophobic and halogenated alkane solutes, less so for those that are more polar or engage in hydrogen bonding (amines, alcohols). But the efficiency of the calculations beats that of all-atom simulations by about an order of magnitude or more.

Why are ion hydration free energies asymmetric with respect to ion charge? Rick Remsing and John Weeks investigate that question using an analytical model for calculating hydration free energies that involves gradually “turning on” the ion-solvent Coulomb interaction (JPCB 120, 6238; 2016 – paper here). This enables them to see why the Born solvation model fails to capture the asymmetry: in short, it works well enough for slowly varying Gaussian charge distributions but not for the abrupt, delta-function-like distributions in ion cores. Only in the latter case is the asymmetry in response to ion charge recovered.

Friday, May 27, 2016

Dewetting and its origins

Several thermophilic proteins are known to have internal water-filled cavities, and some are known to denature when these cavities are emptied. Could this internal water be the secret to their thermal stability? Fabio Sterpone in Paris and his colleagues have investigated that question by using MD simulations to calculate the hydration free energy of buried water for several homologous mesophilic and thermophilic proteins (D. Chakraborty et al., JPCB 119, 12760; 2015 – paper here). Their findings support that main contention: the buried water contributes favourably and significantly to the stability of the thermophilic proteins. This could therefore offer a strategy for designing proteins robust against high temperatures.

Protein folding is studied with proton NMR by Francesco Mallamace, Gene Stanley and their collaborators (F. Mallamace et al., PNAS 113, 3159; 2016 – paper here). In particular, they aim to elucidate the role of water in the folding process, taking lysozyme as the paradigmatic example. They examine the evolution of hydrophilic (amide NH) and hydrophobic (methyl and methine CH) groups as folding proceeds, both for a reversible unfolded intermediate state and the irreversible denatured state. Hydrogen bonding between amide groups and internal water seems to play a crucial role: water acts as a kind of “glue” between buried amide and carbonyl groups, while the increased mobility of hydrophobic groups as they form clusters in the folded state compensates for the loss of configurational entropy that this entails.

Studying the binding and unbinding kinetics of cavity-ligand interactions could offer valuable insights into the efficacy of drugs and drug candidates. But it’s tough to do using MD simulations, because the timescales are often too long. Bruce Berne at Columbia and his colleagues have developed a “metadynamics” scheme that allows MD to probe timescales of not just many seconds but an hour or so (P. Tiwary et al., PNAS 112, 12015; 2015 – paper here). They examine a prototypical ligand–cavity system: a fullerene in a simple hydrophobic pocket, with explicit water. They find that binding involves an abrupt dewetting transition when the fullerene is constrained to move only along the central axis of symmetry of the system, but that dewetting is continuous, and binding is 20-fold shorter-lived (around 200s), when this steric constraint is removed.

Hydration of a fullerene in a simple binding cavity.

Abrupt dewetting has been observed between two planar hydrophobic surfaces at small separations, where it is simply capillary evaporation by another name. But Matej Kanduc and Roland Netz at the Free University of Berlin find that it can also occur between a hydrophobic and mildly hydrophilic surface, if they are both polar but electrically neutral (PNAS 112, 12338; 2015 – paper here). This leads to dry adhesion of the dissimilar surfaces. Kanduc and Netz construct the full phase diagram for any pair of surfaces, showing that dry adhesion can happen even for appreciable hydrophilicity of one surface (say, contact angle of 45 degrees) if the other is markedly hydrophobic (contact angle around 120 degrees).

The phase diagram of attractive/dewetting regimes for two planar surface of different contact angle.

Masataka Nagaoka at Nagoya University and colleagues look at another dewetting process, which takes place in the binding pocket of thrombin when it binds a ligand (I. Kurisaki et al., JPCB 119, 15807; 2015 – paper here). The process is quite complicated. These MD simulations indicate that, following the establishment of a hydrogen-bonding interaction between the substrate and an Asp group in the pocket, the water is gradually removed from the pocket, going not into the bulk phase but into a water channel within the protein – which thus turns out to have a functional role.

The water channel of thrombin.

The importance of density fluctuations at the interface in nanoscale (as opposed to atomic-scale) hydrophobic interactions driven by drying transitions has been asserted for some time now by David Chandler and his colleagues. Phillip Geissler and Suriyanarayanan Vaikuntanathan recently developed this idea by showing that the Lum-Chandler-Weeks drying-induced hydrophobic attraction needs to pay close attention to capillary waves (Phys. Rev. Lett. 112, 020603; 2014). Now they and their coworkers have put this theory to the test in a coarse-grained lattice model (S. Vaikuntanathan et al., PNAS 113, E2224; 2016 – paper here). Despite the relative simplicity of the model, it seems to capture very efficiently the nanoscale density fluctuations and their relevance for hydrophobic hydration in the case of a hydrophobic solute particle at the air-water interface and a pair of nanoscale hydrophobic plates. In both cases the “minimalistic” model agrees very well with detailed atomistic simulations, suggesting that it succeeds in modeling the basic physics of these situations. What seems striking here, I think, is how this long-problematic issue of hydrophobic hydration seems to be turning back towards the “old-style” basic physics of wetting and inhomogeneous fluids (compare, for example, R. Evans & M. C. Stewart, J. Phys. Condens. Matt. 27, 194111; 2015; R. Evans & N. B. Wilding, PRL 115, 016103; 2015).

Another coarse-grained description of hydration is offered by Bill Jorgensen at Yale and colleagues (X. C. Yan et al., JPCB jpcb.6b00399; 2016 – paper here). They use techniques I’m frankly not familiar with to couple a coarse-grained approach with all-atom solute models, and find that the resulting multiscale simulations compare acceptably with the all-atom case while achieving a 7-30-fold reduction in computational cost.

And on the same track, Kenichiro Koga at Okayama and colleagues report a mean-field approximation for inhomogeneous liquids to study hydrophobic hydration, specifically the solvation of methane at the air-water interface (K. Abe et al., JPCB 120, 2012; 2016 – paper here). The approach here is to consider two steps in the solvation process: creation of the cavity for a hydrophobic solute, and insertion of the solute itself.

Ions modify hydrophobic interactions in an ion-specific manner, familiar as Hofmeister effects. But to what extent do these influences depend on the arrangements and mobility of the ions? Izabela Szlufarska at the University of Wisconsin and colleagues examine that question using MD simulations of hydrophobic interactions between a nonpolar surface and nonpolar or amphiphilic nanorods mimicking β-peptides (K. Huang et al., JPCB 119, 13152; 2015 – paper here). They compare the cases of free ions in solution, and ionic groups tethered to the rods both randomly and in organized spatial arrangements (such as a row down one side). Free ions generally strengthen hydrophobic interactions according to the conventional ion-specific series, in a manner that is correlated with (and assumed to be due to) fluctuations in water density close to the surface. But these interactions can be switched on or off by different arrangements of the immobilized ions, and the ion-specific effects now don’t follow the usual Hofmeister ranking. The authors analyse the findings in a dynamical picture, concluding that “Our analysis of the structure and dynamics of water near the hydrophobic nanorod shows that dynamics is a better indicator of the specific ion effect than the static water structure.” Is this perhaps an emerging consensus for such situations?

The nanorods studied by Huang et al.: cyan groups are nonpolar, yellow are immobilized ions.

Hydration water around intrinsically disordered proteins is more mobile than that around globular proteins, according to simulations by Pooja Rani and Parbati Biswas at the University of Delhi (JPCB 119, 13262; 2015 – paper here). They find that both the translational and rotational diffusion are relatively greater for IDPs. In an earlier study, they found that the residence times of hydration waters for IDPs are relatively long (JPCB 119, 10858; 2015) – but that work doesn’t contradict this, they argue, because the enhanced mobility doesn’t actually help the water molecules leave the hydration layer.

Water wires are essential for transporting protons through the pores of the chloride/proton exchanger ClC-ec1, a member of the CLC superfamily of proteins that exchange protons for halides and other anions across cell membranes. That process is elucidated in detail in simulations by Emad Tajkhorshid at UIUC and colleagues (T. Jiang et al., JACS 138, 3066; 2016 – paper here). They examine how the water wires formed in the presence of chloride bound in the central anion binding site differ from those in the presence of fluoride, nitrate and thiocyanate, giving the channel its anion specificity. For fluoride and nitrate there are only “pseudo water wires” separated by the intervening anion, which can’t sustain proton transport; for thiocyanate there are none at all.

Water wires and their disruption/absence for various anions in the channel of the chloride/proton exchanger ClC-ec1.

Is the air-water interface acidic or basic? In other words, do hydrated excess protons or hydroxide ions have an affinity for the water surface? Simulations and experiments have sometimes seemed to give conflicting results, making this a point of controversy. Greg Voth, now at the University of Chicago, and his coworkers reported several years ago that simulations showed protons having an affinity for the interface. They now confirm this finding using state-of-the-art reactive molecular dynamics (Y.-L. S. Tse et al., JACS 137, 12610; 2015 – paper here). The (weak) preference of the hydrated proton of the water surface, they say, is enthalpically favoured and reduces the loss of hydrogen-bonding there. The hydroxide ion, meanwhile, is repelled from the interface, also for enthalpic reasons.

Small peptides don’t sample all of the conformational space theoretically available to them. Brigita Urbanc and colleagues at Drexel University in Philadelphia wonder if hydration structure has something to do with this (D. Meral et al., JPCB 119, 13237; 2015 – paper here). Using MD simulations, they show that the so-called polyproline II-like (pPII) and β-strand conformations that are prominent for GXG peptides (with X a “guest” residue) have different hydration structures, the former being enthalpically stabilized and the latter entropically. In general the nature of backbone hydration in the pPII conformations is clathrate-like, and differs for different X depending on the residue’s ability to template this structure.

It’s looking ever more as though water has an important, even pivotal role in the assembly of the β-amyloid aggregates implicated in neurodegenerative diseases. Nadine Schwierz at UC Berkeley and coworkers report that it drives the growth of these fibrils (N. Schwierz et al., JACS 138, 527; 2016 – paper here). Their simulations suggest that solvent entropy is the main driving force, and that assembly involves collective water motions. As two β strands lock together, motion of the intervening water is significantly retarded, and there is an entropic gain when this water is removed to create a dry binding interface.

The hydrophobic interaction is still challenging to understand intuitively. That’s emphasized by a report by Lawrence Pratt at Tulane University and colleagues showing that, for argon-argon interactions in water, the “hydrophobic bond” is actually weakened by including solute attractive interactions (M. I. Chaudhari et al., JPCB 120, 1864; 2016 – paper here). They treat the situation using local mean field theory, and I think (but am not sure) that ultimately the reason for this result is that in effect the solute attractive interactions benefit solvation more than they do aggregation.

Might water networks in a protein binding site be amenable to a thermodynamic treatment that considers them a kind of loose, composite ligand? That seems to be in essence what Gregory Ross at the University of Southampton and colleagues are attempting to enable (G. A. Ross et al., JACS 137, 14930; 2015 – paper here). They have modified the grand canonical MC simulation technique to allow efficient calculation of the binding energy of an entire hydrogen-bonded water network, as well as to estimate the individual water-molecule affinities and the degree of cooperativity between them. They propose that the method might allow the incorporation of water networks into rational drug design.

How to handle long-ranged forces has, as John Weeks at Maryland and colleagues put it in a paper in PNAS (R. C. Remsing et al., PNAS 113, 2819; 2016 – paper here), “plagued theory and simulation alike”. They offer a solution: a way to incorporate long-ranged interactions that uses only short-ranged potentials. They demonstrate it for the case of hydration of ionic and hydrophobic species. It relies on local molecular field theory, in which the interactions are accommodated by finding a simpler, single-particle “mimic system” that can furnish an effective field that takes care of averaged “far-field” interactions in the full system under study.

Different types of antifreeze proteins have different mechanisms of action, according to Ilja Voets at the Eindhoven University of Technology and colleagues (L. L. C. Olijve et al., PNAS 113, 3740; 2016 – paper here). They have measured both the thermal hysteresis and the ice recrystallization inhibition activity for a range of AFPs, and find no correlation between the two. Both of these properties have been considered to be features of antifreeze activity, which the researchers attribute to binding to different ice planes. They conclude that antifreeze activity is many-factored, and that applications and rational design of AFPs “requires a tailored optimization to the specific purpose.”

More debate on the putative liquid-liquid transition in metastable water: Gyan Johari and José Teixeira argue that the thermodynamic arguments linking this transition to that between low- and high-density amorphous ice don’t stand up (JPCB 119, 14210; 2015 – paper here). They conclude that “The available data show that HDA is not a glass, and the presumed HDL and LDL are not normal liquids. If that is accepted, the HDL−LDL fluctuations view, the two-liquid model, and the virtual liquid−liquid phase transition would all be consequences of a false premise.” I look forward to the next round…

Two quite different forms of metastable (quasi-)liquid water are reported by Gen Sazaki and colleagues at Hokkaido University. They have previously reported such phases on the surface of ice (Sazaki et al., PNAS 109, 1052; 2012); one of the liquid phases wets the ice, the other doesn’t. Using advanced optical microscopy, they now show that these surface phases are kinetically, not thermodynamically, stable, and that they form not by the surface melting of ice but by the deposition of supersaturated water vapour on the ice surface (H. Asakawa et al., PNAS pnas.1521607113; 2016 – paper here).

Julio Fernández continues his studies of “frustrated” water at the protein interface (dehydrons, which are topologically deprived of hydrogen-bonding opportunities) with a study of the acid-base chemistry of such water, using quantum-chemical calculations (FEBS Lett. 590, 215; 2016 – paper here). He reports that water surrounding deydrons is proton-accepting, and enables a mechanism for directed Grotthuss transport of protons

Tryptophan fluorescence has been widely used to study protein dynamics and hydration. Feng Gai and colleagues at the University of Pennsylvania report that a tryptophan analogue, 5-cyanotryptophan can be used in the same way but with greater sensitivity (B. N. Markiewicz et al., JPCB 120, 936; 2016 – paper here). They show that, among other things, the new probe molecule allows them to distinguish two differently hydrated environments in a folded protein (Trp-cage).

Whether or not the result reported by Ruth Livingstone and colleagues at the MPI Mainz is relevant to water in biology isn’t clear, but it’s an interesting finding all the same. They use vibrational spectroscopy to look at water interacting with a monolayer of the detergent sodium dodecyl sulfate at the water surface, and see two distinct types of water present (R. Livingstone et al., JACS 137, 14912; 2015 – paper here). Close to the anionic head groups the waters have localized O-H stretches, but further below the monolayer there is a delocalized O-H mode. These two modes are coupled and can exchange energy. The question is obviously whether the same applies for lipid assemblies in vivo.

Tuesday, March 1, 2016

Why DNA hydration is different from that of proteins

Well, it seems that post-Christmas catching up now takes until March… Hopefully normal service hereafter.

Why trehalose confers protection against urea-induced denaturation of proteins is investigated by Subrata Paul and Sandip Paul of the Indian Institute of Technology in Guwahati using MD simulations for a protein analogue (N-methyl acetamide, NMA) (JPCB 119, 9820; 2015 – paper here). They find that direct interactions are the key. NMA hydration water is displaced by hydrogen-bonded urea, but the addition of trehalose only modestly decreases the urea density close to the amide. Rather, trehalose molecules largely replace water molecules in the hydration shell (so that trehalose and urea bind to NMA simultaneously). For proteins this would reduce the ability of water to solvate exposed backbone in the denatured state.

Another structure-stabilizing osmolyte, trimethylamine N-oxide (TMAO), is studied by Yuki Nagata of the MPI for Polymer Research in Mainz and colleagues (JPCB 119, 10597; 2015 – paper here). Their ab initio simulations look at the effect of TMAO on water reorientational dynamics, and show that these are retarded significantly close to the TMAO’s oxygen atom due to its hydrogen bonding with water – a result not seen using a simpler force-field model that represents the oxygen as a single point charge. Since TMAO is generally excluded from protein surfaces, its stabilizing influence seems in this case to be an indirect effect, for which the authors say this interaction with water seems likely to be important. These conclusions are supported by the work of Gerhard Schwaab and colleagues at Bochum, who have used THz/FIR and Raman spectroscopy to look at the solvation dynamics of TMAO (L. Knake et al., JPCB 119, 13842; 2015 – paper here). They too find strong hydrogen bonding between TMAO and water, which supports an indirect mechanism for its biological effects.

How proteins denature in pure water as a result of low temperature or high/low pressure is still not entirely clear – in particular, are the mechanisms of these two processes essentially the same or distinct? That questions is addressed by Valentino Bianco and Giancarlo Franzese at the University of Barcelona using MC simulations of a coarse-grained protein model (represented as a self-avoiding hydrophobic or mixed hydrophobic/hydrophilic chain) with explicit water solvent (Phys. Rev. Lett. 115, 108101; 2015 – paper here). They argue that both denaturation processes involve a balance of free energies between hydration and bulk water. For cold denaturation, this balance favours the unfolded protein conformation for energetic reasons relating to the increasing stabilizing effect of water-water hydrogen bonds in the hydration shell. High-pressure denaturation, meanwhile, is driven by changes in local water density near the unfolded protein, giving a dominant PV contribution to the free-energy change of denaturation. And low-pressure denaturation is enthalpically driven, again by changes in the number of water-water hydrogen bonds in the hydration shell. In sum, the authors say, “For [all] these mechanisms is essential to take into account how the protein-water interactions affect the stability of the water-water HB and the water density in the hydration shell.”

A strategy for attacking antibiotic resistant bacterial pathogens that has been afforded increasing attention recently involves disabling not the mechanisms by which they synthesize key cellular components but those that contribute to virulence. One such target is a dehydratase enzyme DHQ1, present in several typical “superbugs”. Concepción González-Bello at the University of Santiago de Compostella and coworkers describe an inhibitor containing an ammonium group that binds to DHQ1 without relying on the reactive epoxide functionality of previous such drug candidates (C. González-Bello et al., JACS 137, 9333; 2015 – paper here). It binds to and covalently modifies the enzyme’s active site thanks to hydrogen-bonding to a single water molecule, while a second conserved water molecule participates in the reaction that leads to covalent modification.

Another water-assisted enzymatic mechanism is described by Ana-Nicoleta Bondar of the Free University of Berlin and colleagues. Using MD simulations, they look at the proton-transfer protein PsbO, a subunit of photosystem II, and find low-mobility water molecules close to its surface that form part of a water-carboxylate network that could facilitate proton transport (S. Lorch et al., JPCB 119, 12172; 2015 – paper here). Some of these waters might, they say, also assist the docking of PsbO into the PSII complex.

The water-carboxylate network on the surface of the proton-transfer protein PsbO.

In contrast to the often slow dynamics of “functional water” at protein surfaces, Songi Han at UCSB and colleagues report anomalously fast water diffusion near DNA surfaces (J. M. Franck et al., JACS 137, 12013; 2015 – paper here). They use Oberhauser effect dynamic nuclear polarization (ODNP) to look at water translation dynamics along the backbone, which entails adding spin centres along a DNA backbone to act as “reporters”. Anomalously fast here means bulk-like, in contrast to the retarded diffusion of most of the hydration water, suggesting that the DNA-water interactions are only weak for a significant proportion of the hydration shell. Such water is relatively easily displaced when proteins bind to the DNA. Thus, in comparison to proteins, DNA would be capable of storing less free energy that can be released entropically in binding interactions – and, the authors argue, makes DNA duplexes less specific in their interactions (outside, of course, of the specificity supplied by the sequence itself). So “the high mobility of the solvation water around DNA may have been tailored to its role in biological function.”

I had the pleasure of meeting Xiao Cheng Zeng of the University of Nebraska in Shanghai in November, and hearing about his interesting work on monolayer and bilayer ices. He and coworkers have recently explored the mechanism by which a preference for potassium over sodium ion transport arises in synthetic hydrophobic organic nanopores, namely the stacked macrocycles reported by Zhou et al. in Nat. Commun. 3, 1-8 (2012), and compare them with carbon nanotubes (H. Li et al., PNAS 112, 108512; 2015 – paper here). At face value the preferential K+ transport is surprising, given that K+ has the larger hydration sphere. But the researchers explain this selectivity (which is also seen in some biological ion channels) in terms of the greater robustness and structure of the Na+ hydration shell. Moreover, the roughness of the interior surface of the organic nanopores lowers the diffusivity of both ions – but more so sodium – relative to smooth-sided carbon nanotubes, with the result that the CNTs offer the highest rate of selective K+ transportation.

Jianzhong Wu at the University of California at Riverside use a combination of thermodynamic measurements (Henry’s constants) and MD simulations to examine how the degree of hydrogen bonding among water molecules changes in hydrophobic hydration shells (J. Kim et al., JPCB 119, 12108; 2015 – paper here). They find that the extent of H-bonding is slightly reduced, and that this is a significant factor in the positive hydrophobic hydration heat capacity. Certainly nothing ice- or clathrate-like here.

How do we assess the hydrophobicity of heterogeneous surfaces, composed of a mixture of hydrophobic and hydrophilic patches? Well, it’s complicated. Damián Scherlis at the University of Buenos Aires and colleagues find in simulations with coarse-grained mW water that there is a rather complex variation of water-droplet contact angle on such surfaces with the amount, distribution and arrangement of hydrophilic (hydrogen-bonding) domains on a hydrophobic surface (M. H. Factorovich et al., JACS 137, 10618; 2015 – paper here). In this case, there may be a nonlinear variation of contact angle with composition, the angle being largest for a particular fraction of hydrophobic species (about 0.6 when randomly mixed). In other words, this mixture can have a larger contact angle than the purely hydrophobic surface. What’s more, this variation depends on the size of the domains: the nonlinearity tends to vanish for hydrophilic domains bigger than about 2 nm, when the behaviour returns to being reasonably well described by the Cassie model. The same conclusions are supported by looking at the desorption pressure of water in nanopores of these compositions. This appears to offer considerable scope for tuning the wettability of surfaces.

And I have just discovered that a paper to which I contributed in a very small way, on the notion of chaotropicity as a way of understanding the limiting tolerances of biofuel-producing microorganisms, was published at the end of last year (J. A. Cray et al., Curr. Opin. Biotechnol. 33, 228; 2015 – paper here). My coauthors are to be credited with everything of real substance in this interesting and potentially useful article.