Francesco Mallamace at MIT/Messina and colleagues have used proton NMR to follow the rearrangements of water during the thermal (heat and cold) denaturation of lysozyme (F. Mallamace et al., J. Phys. Chem. B 115, 14280; 2011 – paper here). Since the chemical shift is considered to probe hydrogen-bonding interactions in the water network, tracking it as the temperature changes take the protein from the folded to the denatured states reveal some aspects of how changes in protein structure are mirrored by those of the hydration water. The researchers conclude that water plays an active role in the process, and that as denaturation proceeds, the average number of H-bonds with which each water molecule is involved changes correspondingly.
How glycerol acts as a cryoprotectant is the subject of a study by J. Towey and L. Dougan at Leeds (J. Phys. Chem. B jp2093862 - paper here). They use neutron scattering to investigate whether, as one hypothesis has it, dissolved glycerol perturbs water structure to modify the hydrogen bonding ability and suppress ice formation. The solution turns out to be well mixed, with many glycerol monomers, but there is no discernible perturbation of the first coordination shell of water. The second shell, however, is perturbed in a manner similar to that caused by elevated pressure. Quite what this means for “the ability of water molecules to form ice” isn’t clear (indeed, I’m not even too sure what that expression means – displacement of the phase boundary?), but Towey and Dougan conclude that any explanation will need to focus not simply on local perturbations (or changes to water’s ability to form hydrogen bonds) but on changes to the extended hydrogen-bonded network.
More on the mechanisms of osmolytes: Dave Thirumalai and colleagues at Maryland have studied the stabilization of compact peptide conformations by trimethylamine N-oxide (TMAO) (S. S. Cho et al., J. Phys. Chem. B 115, 13401; 2011 – paper here). They attribute it to direct interactions between TMAO and the protein surface, which exclude solvent there. This is then a kind of excluded-volume effect analogous to the stabilization of proteins by molecular crowding (in which there is entropic destabilization of the unfolded state) – making TMAO a ‘nanocrowding’ particle.
In a related vein, M. Paulaitis at Ohio State and colleagues describe an integral-equation approach to deduce preferential interactions between cosolvents, which they say can be used to deduce preferential interactions of cosolvents such as osmolytes, naturants and cryoprotectants locally on the surface of proteins (M. H. Priya et al., J. Chem. Phys. B 115, 13633; 2011 – paper here).
Personally, I find it hard to think about hydration in crowded environments, in which marcomolecules might disturb one another’s hydration shells and sometimes temporarily associate with one another in non-specific ways. Sergio Hassan and Peter Steinbach at NIH have tried to provide a context from framing this question (J. Phys. Chem. B 115, 14668; 2011 – paper here). One issue is how incomplete and anisotropic hydration might create electrostatic effects. Another is how solvation forces due to structured hydration shells (layering, for example) manifest themselves on hydrogen-bonding at solute-water interfaces. Using a continuum solvent model, they say that the electrostatic effects of solvent exclusion can have a strong impact on protein-protein binding. But I think it fair to say that at this point the paper is largely presenting the methodology for investigating the problem, rather than reaching general conclusions about how these aspects of crowding affect the molecular biology.
Confinement will, of course, do other things to water itself. Ivan Brovchenko and Alla Oleinikova have predicted that water in slit-like pores just 2.4 nm wide might undergo the liquid-liquid transition predicted in the metastable bulk at low temperature and high pressure (J. Chem. Phys. 126, 214701; 2007). Limei Xu and Valeria Molinero at Utah have now examined that idea in simulations using their mW model of water held within 1.5-nm diameter cylindrical pores (J. Phys. Chem. B 115, 14210; 2011 – paper here). This system is comparable to the pores of MCM-41 nanoporous silica, as used in recent experiments on confined water (e.g. L. Liu et al., Phys. Rev. Lett. 95, 117802 (2005)). They find no evidence for a first-order liquid-liquid transition, but note that smearing of discontinuous transitions is well known in pores (although not in fact inevitable), and therefore that this doesn’t rule out the existence of such a transition in the bulk. For their simulations of the bulk phase, they do see a possible signature of a L-L critical point – a locus of maximum compressibility – but can’t study this in detail because fast crystallization makes it impossible to equilibrate a metastable water phase in this region.
How do proteins remain dynamic while remaining soluble and resistant to aggregation? Fabrizio Chiti at the University of Florence, Chris Dobson at Cambridge, and their colleagues, have sought to answer this by combining NMR relaxation data, H/D exchange experiments and MD simulations (A. De Simone et al., PNAS 108, 21057; 2011 – paper here). They use the fruitfly acylphosphatase as their model system, and find that the wild-type protein has free-energy barriers that limit access to aggregation-prone conformations except under aggrtegation-prone conditions (addition of small amounts of trifluoroethanol). They sum up the situation nicely: “The sensitivity of the energy surfaces of proteins to minor perturbations supports the view that there is a delicate balance between functionality, stability, and solubility, which is encapsulated by the concept of ‘life on the edge’”.
Hydrogen bonds hydrating hydrophobic regions of a protein or peptide seem to have a greater strength than those in bulk water. It’s been suggested that this might be not so much because the H-bonds are genuinely strengthened but because the orientational preferences of H-bonds in such a situation result in a depletion of weaker, strained H-bonds, i.e. a change in the population (Zichi & Rossky, J. Chem. Phys. 83, 797 (1985)). Peter Rossky and colleagues have now explored this idea further using MD simulations of a 16-residue peptide (J. Phys. Chem. B 115, 14859; 2011 – paper here). They find support for the idea, namely, water is depleted of near neighbours around apolar groups and so samples lower-coordination configurations that are undistorted and unstrained. In other words, the phenomenon is primarly a kind of packing effect.
A new angle on Hofmeister effects is offered by Huib Bakker at FOM Institute for Atomic and Molecular Physics in Amsterdam, who have looked at the orientational dynamics of water around various ions (K. J. Tielrooij et al., J. Chem. Phys. B 115, 12638; 2011 – paper here). When the salt consists of a strongly hydrated ion and a weakly hydrated counterion, the water molecules hydrating the former have impeded orientational dynamics, making it strongly anisotropic. In that case, they say that hydration is ‘semi-rigid’ in the first hydration shell: affected along one vector but not along others. If both ions are strongly hydrated, such perturbations of water dynamics extend well beyond the first hydration shell.
And while we’re there: Daryl Eggers and colleagues at San José State University have taken a thermodynamic line of attack on Hofmeister by determining the molar water volumes in various concentrated electrolyte solutions (A. Y. Payumo et al., J. Phys. Chem. B 115, 14784; 2011 – paper here). They find that the solutions are highly nonideal, presumably because of strong competition under these conditions for hydration water. Moreover, the solubility of the small amide diketopiperazine follows the Hofmeister series for all the anions and cations studied, and the authors explain this on the basis that Hofmeister effects are governed by changes in the average free energy of the bulk aqueous phase – that is, if Hofmeister effects are a bulk phenomenon of water.
The tale of the low-temperature dynamical crossover for hydrated proteins and their hydration shells continues to get more complicated. Using dielectric spectroscopy and MC simulations, Gene Stanley, Giancarlo Franzese and their colleagues now report evidence of two such crossovers for the hydration water of lysozyme: one at about 252 K, the other around 181 K (M. G. Mazza et al., PNAS 108, 19873; 2011 – paper here). Marie-Claire Bellissent-Funel and her colleagues have previously seen something similar – two transitions at 220 and 150 K (J.-M. Zanotti et al., PCCP 10, 4865; 2008). Stanley et al. now ascribe the first of these to maximal fluctuations in the making and breaking of hydrogen bonds, and the second to maximal fluctuations in cooperative reordering of the H-bonded network.
Meanwhile, Sol Gruner and colleagues at Cornell report evidence for another protein dynamical transition right down at 110 K, which they say correlates with the transition of the hydration water from a high- to a low-density amorphous state (C. U. Kim et al., PNAS 108, 20897; 2011 – paper here).
Wilfred van Gunsterden at ETH and colleagues show how solvation free energies, as well as the free energies of protein-ligand binding and protein conformational dynamics, can be calculated using a new software package called GROMOS, which van Gunsterden and colleagues have introduced in a paper in press with Comput. Phys. Commun. (S. Riniker et al., J. Phys. Chem. B 115, 13570; 2011 – paper here).
Alan Soper has an intriguing paper in J. Phys. Chem. B (115, 14014; 2011 – paper here) in which he presents a new mixture model of water. This isn’t exactly a two-state model – the two forms are intimately mixed – but it postulates two populations of water molecules, each of which can form hydrogen bonds only with molecules of the other type and not with those of their own type. This is not, as far as I can see, intended as a literal representation of some molecular-scale distinction – both types of water molecule have identical structure – but is imposed as a device for introducing a three-body term into the interactions. The results show good agreement with structural studies using neutron and X-ray scattering, and give rise to a situation where water molecules are H-bonded to some of their neighbours but not others. This, Alan suggests, is perhaps why mixture models of water have been so enduring: not because there really are two distinct populations but because – if I’m understanding this correctly – the three-body terms have the effect of making it appear that way.
Michele Parrinello and colleagues at ETH have investigated the recombination of hydronium and hydroxide ions in water using ab initio MD simulations (A. Hassanali et al., PNAS 108, 20410; 2011 – paper here). They find that the mechanism is rather different from what has traditionally been assumed in terms of a Grotthuss mechanism. The researchers say that the Grotthuss mechanism serves to bring the hydronium and hydroxide to a distance of around 6 Å, when they are bridged by two water molecules as a ‘water wire’. But then there is a collective compression of this water wire that results in a concerted motion of three protons (rather than a series of distinct one-proton hops), converting both ions into water molecules.
Thursday, January 19, 2012
Tuesday, January 10, 2012
Welcome to 2012
Lots to catch up on here, and while I’m not going to do that exhaustively now, here is an update to let you know that this blog will still be active in 2012.
I’ve recently written a News & Views article for Nature (478, 467; 2011 – here) about, among other things, the recent paper by George Whitesides’ group (PNAS 108, 17889; 2011 – paper here) on the hydrophobic effect in ligand binding – which, as discussed in the previous post, suggests that there is not any single ‘hydrophobic effect’ operating here, but a delicate and case-specific balance of enthalpic and entropic effects. They noted that in the example they studied, the ‘hydrophobic’ aspect of binding was driven largely by the enthalpic effect of displacing/rearranging water around nonpolar contacts. That notion is supported by a paper by Stephen Martin and colleages at the University of Texas at Austin, who find something rather similar for small peptide binding by the SH2 domain of the growth receptor binding protein Grb2 (J. M. Myslinski et al., JACS 133, 18518; 2011 – paper here). They say that increases in the nonpolar contact area don’t necessarily lead to entropic gains due to release of water, but have a primarily enthalpic influence on binding affinity.
How quickly drugs bind to their target molecules can have important pharmacological implications, but these kinetics are generally poorly understood, and thus to design rationally. Xavier Barril from the University of Barcelona and colleagues show using MD simulations that slow kinetics are often a consequence of the presence of buried polar atoms which form hydrogen bonds that are shielded from water, because of the slowness of dehydration/rehydration (P. Schmidtke et al., JACS ja207494u – paper here). In other words, the pace of events in binding can be set by the degree of water accessibility. They show that this effect can be predicted from structural data, and can thus potentially be accessible to design.
Knowing the hydration structure of proteins is important for small-/wide-angle X-ray scattering (SWAXS) studies of proteins not so much for its own sake but because the contribution of the solvent to the scattering must be subtracted in order to extract information about the protein secondary structure. It’s easy enough to extract the contribution of bulk water, but correcting for the scattering of the hydration shell is complicated. Tobin Sosnick and coworkers at the University of Chicago now show two ways to do this (J. J. Virtanen et al., Biophys. J. 101, 2061-2069; 2011 – paper here). One is to deduce the solvation structure by full MD simulations, which they say gives results that match the SWAXS data closely. But they have also developed a much more computationally less intense solvation model called HyPred, which gives a scattering profile that agrees well both with MD and with experiment.
Bernhardt Trout and his coworkers at MIT present an illustration of just how complex the interactons of proteins and ions can be (D. Shukla et al., JACS ja205215t – paper here). Complex ions such as guanidinium create a particularly intricate picture. Gdm+ can destabilize proteins via the formation of hydrogen bonds and electrostatic interactions, but when paired with an anion that is a hydrogen-bonding acceptor it can form clusters with the ions, which suppresses the effect. Molecules (such as arginine) with multiple Gdm+ groups are sometimes used to suppress protein aggregation, and can do so without compromising protein stability. Trout and colleagues investigate the effect of arginine oligomers (n=1-4) on protein aggregation and conformational stability for two different anions, chloride and sulphate. While monomeric arginine chloride is used as an aggregation suppressor, the n-mers only inhibit aggregation at low concentration – they actually accelerate it at moderate to high concentration. Meanwhile, the sulphates inhibit aggregation at all concentrations. And while the chlorides reduce protein stability, the sulphates enhance it. The researchers explain all this in terms of the balance between ion-ion and ion-protein interactions.
All this mirrors an increasing tendency to consider Hofmeister effects of ions on proteins in terms of direct interactions between the two species, rather than as indirect consequences of changes in hydration. Elena Algaer and Nico van der Vegt at the TU Darmstadt provide some support for this notion with a study of the salting-in and –out of small model amides by various sodium salts (J. Phys. Chem. B jp208583w – paper here). They say, for example, that the salting-in if NiPAM by NaI is mediated by interactions of iodide with the nonpolar groups. Such interactions also explain why, of all the salts studied, the iodide alone fails to induce hydrophobic collapse of polyNiPAM.
How does water permeate cell membranes? Its passage through the water-regulating membrane protein aquaporin is fairly well studied, but little is known about other water transporters. The bacterial sodium-galactose transporter vSGLT and its human homologue the sodium-glucose cotransporter hSGLT1 both have the potential to let water through. Jean-Yves Lapointe at the Université de Montréal and colleagues have used MC and MD simulations to show that indeed these protein pores can be filled with water (a pathway of about 100 molecules) which allows passive water permeation (L. J. Sasseville et al., Biophys. J. 101, 1887-1895; 2011 – paper here). This pathway depends on the proteins’ conformation: there is a constriction at one point which reduces the water bridge to a single-molecule chain which then ‘snaps’ at a ‘hydrophobic plug’, creating a 4.3 Å gap of low water density. But the resulting barrier to water permeation is conformation-dependent, and can be altered by varying the membrane potential. The passage of a sugar molecule also can bring water with it, but the mechanism of this is still open to debate.
Jhih-Wei Chu and colleagues at Berkeley conclude from MD simulations that the insolubility of cellulose in water is an entropic effect due primarily to the reduction of solvent entropy if the glucan chains in a fibril unravel (A. S. Gross et al., J. Phys. Chem. B 115, 13433; 2011 – paper here). There are some lessons here for how to solubilize cellulose in other solvents, such as ionic liquids.
Why and how do ions segregate at the air-water interface? This phenomenon seems well attested, with anions, especially large and polarisable ones, tending to accumulate at the interface. But the reason for this is still debated. Yi Qin Gao if the Beijing National Laboratory for Molecular Sciences and coworkers investigate the question with MD simulations, looking in particular at the differences in how anions and cations are solvated (L. Yang et al., J. Phys. Chem. B jp207652h – paper here). They argue that these differences are due to the charge distribution in the water molecules themselves, and that water can approach anions more closely than cations. They suggest that these differences in hydration account for why anions tend to populate the interface more readily.
And on the same topic, Pavel Jungwirth and colleagues have looked at how the hydration of guanidinium ions affects their orientation at the air-water interface (E. Wernersson et al., J. Phys. Chem. B jp207499s – paper here). Guanidinium is depleted at the interface, but the ions that do stay there are preferentially oriented parallel to the surface: in this configuration, it can sit at the surface without needing to break hydrogen bonds. Another way of looking at this is that the ions can take advantage of the deficit of hydrogen bonds between waters at the surface. The authors suggest that similar reasoning might account for the unexpected orientation of some arginine groups (with an analogous structure) in protein side chains.
I’ve recently written a News & Views article for Nature (478, 467; 2011 – here) about, among other things, the recent paper by George Whitesides’ group (PNAS 108, 17889; 2011 – paper here) on the hydrophobic effect in ligand binding – which, as discussed in the previous post, suggests that there is not any single ‘hydrophobic effect’ operating here, but a delicate and case-specific balance of enthalpic and entropic effects. They noted that in the example they studied, the ‘hydrophobic’ aspect of binding was driven largely by the enthalpic effect of displacing/rearranging water around nonpolar contacts. That notion is supported by a paper by Stephen Martin and colleages at the University of Texas at Austin, who find something rather similar for small peptide binding by the SH2 domain of the growth receptor binding protein Grb2 (J. M. Myslinski et al., JACS 133, 18518; 2011 – paper here). They say that increases in the nonpolar contact area don’t necessarily lead to entropic gains due to release of water, but have a primarily enthalpic influence on binding affinity.
How quickly drugs bind to their target molecules can have important pharmacological implications, but these kinetics are generally poorly understood, and thus to design rationally. Xavier Barril from the University of Barcelona and colleagues show using MD simulations that slow kinetics are often a consequence of the presence of buried polar atoms which form hydrogen bonds that are shielded from water, because of the slowness of dehydration/rehydration (P. Schmidtke et al., JACS ja207494u – paper here). In other words, the pace of events in binding can be set by the degree of water accessibility. They show that this effect can be predicted from structural data, and can thus potentially be accessible to design.
Knowing the hydration structure of proteins is important for small-/wide-angle X-ray scattering (SWAXS) studies of proteins not so much for its own sake but because the contribution of the solvent to the scattering must be subtracted in order to extract information about the protein secondary structure. It’s easy enough to extract the contribution of bulk water, but correcting for the scattering of the hydration shell is complicated. Tobin Sosnick and coworkers at the University of Chicago now show two ways to do this (J. J. Virtanen et al., Biophys. J. 101, 2061-2069; 2011 – paper here). One is to deduce the solvation structure by full MD simulations, which they say gives results that match the SWAXS data closely. But they have also developed a much more computationally less intense solvation model called HyPred, which gives a scattering profile that agrees well both with MD and with experiment.
Bernhardt Trout and his coworkers at MIT present an illustration of just how complex the interactons of proteins and ions can be (D. Shukla et al., JACS ja205215t – paper here). Complex ions such as guanidinium create a particularly intricate picture. Gdm+ can destabilize proteins via the formation of hydrogen bonds and electrostatic interactions, but when paired with an anion that is a hydrogen-bonding acceptor it can form clusters with the ions, which suppresses the effect. Molecules (such as arginine) with multiple Gdm+ groups are sometimes used to suppress protein aggregation, and can do so without compromising protein stability. Trout and colleagues investigate the effect of arginine oligomers (n=1-4) on protein aggregation and conformational stability for two different anions, chloride and sulphate. While monomeric arginine chloride is used as an aggregation suppressor, the n-mers only inhibit aggregation at low concentration – they actually accelerate it at moderate to high concentration. Meanwhile, the sulphates inhibit aggregation at all concentrations. And while the chlorides reduce protein stability, the sulphates enhance it. The researchers explain all this in terms of the balance between ion-ion and ion-protein interactions.
All this mirrors an increasing tendency to consider Hofmeister effects of ions on proteins in terms of direct interactions between the two species, rather than as indirect consequences of changes in hydration. Elena Algaer and Nico van der Vegt at the TU Darmstadt provide some support for this notion with a study of the salting-in and –out of small model amides by various sodium salts (J. Phys. Chem. B jp208583w – paper here). They say, for example, that the salting-in if NiPAM by NaI is mediated by interactions of iodide with the nonpolar groups. Such interactions also explain why, of all the salts studied, the iodide alone fails to induce hydrophobic collapse of polyNiPAM.
How does water permeate cell membranes? Its passage through the water-regulating membrane protein aquaporin is fairly well studied, but little is known about other water transporters. The bacterial sodium-galactose transporter vSGLT and its human homologue the sodium-glucose cotransporter hSGLT1 both have the potential to let water through. Jean-Yves Lapointe at the Université de Montréal and colleagues have used MC and MD simulations to show that indeed these protein pores can be filled with water (a pathway of about 100 molecules) which allows passive water permeation (L. J. Sasseville et al., Biophys. J. 101, 1887-1895; 2011 – paper here). This pathway depends on the proteins’ conformation: there is a constriction at one point which reduces the water bridge to a single-molecule chain which then ‘snaps’ at a ‘hydrophobic plug’, creating a 4.3 Å gap of low water density. But the resulting barrier to water permeation is conformation-dependent, and can be altered by varying the membrane potential. The passage of a sugar molecule also can bring water with it, but the mechanism of this is still open to debate.
Jhih-Wei Chu and colleagues at Berkeley conclude from MD simulations that the insolubility of cellulose in water is an entropic effect due primarily to the reduction of solvent entropy if the glucan chains in a fibril unravel (A. S. Gross et al., J. Phys. Chem. B 115, 13433; 2011 – paper here). There are some lessons here for how to solubilize cellulose in other solvents, such as ionic liquids.
Why and how do ions segregate at the air-water interface? This phenomenon seems well attested, with anions, especially large and polarisable ones, tending to accumulate at the interface. But the reason for this is still debated. Yi Qin Gao if the Beijing National Laboratory for Molecular Sciences and coworkers investigate the question with MD simulations, looking in particular at the differences in how anions and cations are solvated (L. Yang et al., J. Phys. Chem. B jp207652h – paper here). They argue that these differences are due to the charge distribution in the water molecules themselves, and that water can approach anions more closely than cations. They suggest that these differences in hydration account for why anions tend to populate the interface more readily.
And on the same topic, Pavel Jungwirth and colleagues have looked at how the hydration of guanidinium ions affects their orientation at the air-water interface (E. Wernersson et al., J. Phys. Chem. B jp207499s – paper here). Guanidinium is depleted at the interface, but the ions that do stay there are preferentially oriented parallel to the surface: in this configuration, it can sit at the surface without needing to break hydrogen bonds. Another way of looking at this is that the ions can take advantage of the deficit of hydrogen bonds between waters at the surface. The authors suggest that similar reasoning might account for the unexpected orientation of some arginine groups (with an analogous structure) in protein side chains.
Thursday, October 13, 2011
Lum-Chandler-Weeks under the microscope
Shekhar Garde and his colleagues have shown in several recent publications how hydration of solutes and its consequences, such as conformational changes in polymers, can be significantly altered near the air-water interface. The more general effect of interfaces on hydrophobic phenomena is now probed by Garde along with David Chandler, Amish Patel and others (A. Patel et al., PNAS doi 10.1073/pnas.1110703108 – paper here). They look at how interactions between hydrophobic solutes of various sizes from sub-nm to several nm are altered close to the interface of water with self-assembled monolayers of various surface chemistries, from hydrophobic to hydrophilic. They find that the driving force for the assembly of hydrophobic particles is smaller near a hydrophobic surface than it is in bulk, and decreases with increasing temperature, in contrast to the bulk (and to hydrophilic surfaces). This implies that hydrophobic surfaces should act as catalysts for the unfolding of proteins. It might also account for how chaperonins work: their initially hydrophobic surfaces help misfolded proteins to unfold, and then ATP-driven conversion of the walls to hydrophilic release the unfolded protein from the wall so that it might fold again in bulk.
Misfolding in the context of Lum-Chandler-Weeks theory is also the subject of a paper by Ruhong Zhou and colleagues (Z. Yang et al., J. Phys. Chem. B 115, 11137 (2011) – paper here). They have looked at whether dewetting transtions play a role in the assembly of two amyloidogenic beta-sheets. For the peptides considered there are two conformations that lead to stacking of beta-sheet pairs: one containing water between the sheets in a 2D slab-like geometry, the other with the water in a 1D tube-like geometry. In both cases the sheets are brought together by a drying transition, but surprisingly this is stronger for the slab-like than the tube-like case. This is attributed here to the different surface roughness of the two packing modes: the ‘staggered’ packing in the slab-like case, which is rougher, disrupts the H-bonding network of the intervening water to a greater extent.
Some further new insights into the Lum-Chandler-Weeks dewetting mechanism for hydrophobic interactions are offered by two recent papers. Li and Walker appear to see the cross-over region between the mechanisms of hydrophobic hydration at small (<1 nm) and large (>1 nm) scales that this theory predicts, by measuring the free energy of hydration of individual monomers of various size in hydrophobic polymers, using an AFM to pull the chains out from a collapsed conformation (Li, I. T. S. & Walker, G. C. PNAS 108, 16527-16532; 2011 – paper here). At a monomer size of around 1 nm in the temperature region of 50 C or so, they find a switch in the hydration entropy from negative to positive (this crossover size reduces to just 3.5 Å at 150 C). Shekhar Garde and Amish Patel have published a commentary on it (10.1073/pnas.1113256108).
And Garde and Patel have joined forces with David Chandler and others in a preprint (arxiv.org/1109.4431 – paper here) that attempts to unravel the issue of why some protein subunits (such as melittin) seem to aggregate via dewetting while others (such as BphC) do not. They find that at model (SAM) hydrophobic surfaces, simulations show that the statistics of large-amplitude fluctuations in the density of interfacial water are altered relative to the Gaussian stats of both bulk water and water at a hydrophilic surface. In other words, despite similar average interfacial water densities, the fluctuations reveal the proximity of the hydrophobic interface to a dewetting transition. This tuning of the interfacial water is, they argue, common to biological systems, where it induces a strong sensitivity to small changes in conformation, allowing the system to take advantage of the phase transition in engineering biomolecular function (in a manner analogous to the finely balanced wetting or drying of ion channels for ‘vapour-lock’ gating). Although melittin and BphC lie on opposite sides of this transition, small modifications to both can tip the balance one way or the other. This offers what seems to me to be a persuasive argument that dewetting is relevant to hydrophobic aggregation even if it does not exactly provide the mechanism for it in all (or even in most) cases: the transition is, if you like, ‘there’ even if it doesn’t manifest itself.
Alenka Luzar and her colleagues have considered this issue from the perspective of whether or not cavitation can take place at the protein-protein interface (J. Wang et al., PCCP 10.1039/c1cp22082a – paper here). They present a lattice model which offers a fast method for predicting if cavitation can happen, and find that part of the surface of melittin is sufficiently hydrophobic to permit this on a timescale that is consistent with that seen in the earlier simulations.
Alenka and her colleagues have also examined how this putative crossover length-scale for hydration behaviour is influenced by charge on the solute (J. Stat. Phys. 10.1007/s10955-011-0337-1 – paper here). They conclude that, for moderate charge, the electrostatic contribution to the solvation free energy is in fact essentially independent of solute curvature, because of a compensation between counterion shielding and the dielectric screening of water – the solvation free energy remains more or less a function only of solute surface area.
To what extent protein-ligand binding requires an atomistic description of changes in hydration is a crucial question, not least for attempts to design synthetic ligands in drug development. Ulf Ryde at Lund University and colleagues have looked at this issue by comparing the ability of continuum methods to predict binding free energies for four different protein-ligand pairs with quite different degrees of solvent exposure at the binding site (S. Genheden et al., JACS ja202972m – paper here). They find that the continuum methods often perform badly, particularly for cases with a greater degree of solvent exposure. We need to know precisely where the waters are and where they go.
And that is somewhat elucidated by Nan-jie Deng at Rutgers University and colleagues for the case of two synthetic inhibitors of HIV-1 protease, Nelfinavir and Amprenavir (N.-j. Deng et al., J. Phys. Chem. B jp204047b – paper here). The binding of these drugs is apparently entropically driven, but the question is where that entropic contribution comes from. A classical view would be inclined to attribute it to the release of water from the binding cleft, but it seems that this isn’t so: these MD simulations suggest that any entropy gain there is more than offset by the restriction of ligand rotation and vibration on binding. Instead, the favourable entropic contribution seems to come from desolvation of the ligand.
A different view of water’s influence on bimolecular recognition is provided by Stacey Wetmore and colleagues at the University of Lethbridge in Alberta (F. M. V. Leavens et al., J. Phys. Chem. B jp205424z – paper here). They have looked at how water molecules can affect the pi-pi interactions between DNA and DNA-binding proteins. Solvating water molecules seem to have essentially no influence on the strength of pi-pi stacking for histidine and adenine, but do weaken the interaction if the histidine is protonated. The latter interaction, however, remains in all cases stronger than the former.
Protons seem to be delivered to membrane proteins such as the proton pump cytochrome c oxidase via some kind of surface-enhanced, two-dimensional transport at the membrane surface. This has been previously postulated as a series of jumps between ionisable groups (phosphate and carbonyl) at the membrane surface. But that notion is challenged by Peter Pohl at the Johannes Kepler University in Linz and colleagues, whose fluorescence measurements of proton transfer at membranes show that proton transport can be equally fast in the absence of ionisable groups (A. Springer et al., PNAS 108, 14461-14466; 2011 – paper here). They conclude that it is probably the network of interfacial water molecules that is responsible instead for the rapid proton motion: as they say, “water structuring at the interface seems to be mandatory for providing the pathway”.
It has of course been long thought that protons may be delivered to the interior of an enzyme via chains of water molecules. That process is studied by heme peroxidase by Emma Lloyd Raven and coworkers at the University of Leicester (I. Efimov et al., JACS ja2007017 – paper here). Kinetic isotope effects reveal that the proton pathway utilizes a Grotthus-like shuttling of protons along a pathway towards the ferryl oxygen that involves three bound waters and two arginine residues.
A model system for studying such proton transfer in confined geometry is reported by Bradley Habenicht and Stephen Paddison at the University of Tennessee in Knoxville (J. Phys. Chem. B jp205787f – paper here). They use MD simulations to look at how protons are transported within carbon nanotubes whose inner walls are functionalized with perfluoro sulphonic acid groups. If these groups are spaced far apart (~8 Å), they tend to be individually hydrated by clusters of water molecules with little interaction between them, and correspondingly reduced acidic proton dissociation. But there is also a pronounced effect of confinement at small nanotube diameters: in the smaller tubes there are stronger interactions between the walls and the water molecules, which can lead to break-up of the hydrogen-bonded network of waters linking the sulphonic acid groups. That network may be restored by polarized charges of fluorine atoms attached to the nanotube walls.
ATP hydrolysis in the active cleft of actin plays an important role in the state of its filamentous form F-actin, affecting its rigidity and its binding of regulatory proteins. There is water in this active site, but it hasn’t previously been clear what, if anything, it does. Marissa Saunders and Greg Voth at Chicago have clarified this through MS simulations based on the crystal structure (J. Mol. Biol. 413, 279-291; 2011 – paper here). They say that the ordered waters help the protein to flatten and brings about a conformational change that promotes ATP hydrolysis. These changes also stabilize the charge on the phosphate and accelerate the deprotonation of the catalytic water involved in hydrolysis. In short, the bound water helps to organize the active-site geometry.
Another nail in the coffin of ‘structure-making and –breaking’: Fabio Bruni and colleagues in Rome have looked at how local solvent structure around ions affects their influence on viscosity – specifically, on how changes in ionic concentration affect the viscosity of the solution (T. Corridoni et al., J. Phys. Chem. B jp202755u – paper here). Classically, the viscosity is found to be (almost) linearly related to the concentration. It has been asserted that the magnitude of the coefficient of proportionality, denoted B, depends on how the ions perturb the water structure. Fabio et al. use neutron scattering and simulations to look for some structural parameter that can be correlated with B. They find that the nature of the (univalent) ions is all but irrelevant to the size of the percolating water clusters in solution. The change in viscosity seems to be unrelated to any structural changes in the bulk liquid, but instead pertain to changes in the local hydration shells of the ions. As a result, they say, “the particular effect of solutes ranked in the Hofmeister series must be looked at in terms of specific ion interactions with hydrophilic or hydrophobic surfaces”, and not in terms of any generalized propensity for structure-making or –breaking.
In an intriguing preprint, Jampa Maruthi Pradeep Kanth and Ramesh Anishetty at the Institute of Mathematical Sciences in Chennai propose that the hydrophobic interaction should be understood as an effect analogous to the Casimir effect (the attraction of two surfaces separated by a vacuum due to the suppression of long-wavelength electromagnetic fluctuations of the vacuum in the gap) (preprint here). Their molecular mean-field analytical method suggests that confinement alters the allowed fluctuations of the hydrogen-bond network, specifically the long-ranged correlations between water molecular orientations. Now what I’d like to know is whether an explicit connection can be made to the alleged role of fluctuations in the Lum-Chandler-Weeks model. But that seems to argue in the opposite direction, namely that fluctuations are actually enhanced in the gap owing to the destabilizing influence of the hydrophobic surfaces on the intervening water layer. It’s not clear to me whether in Kanth and Anishetty the water in the gap is, aside from the suppression of fluctuations, any different from bulk water, except perhaps for the monolayer adjacent to the surfaces…? And why would this not work for hydrophilic confinement too?
Alenka Luzar and her colleagues Christopher Daub and Dusan Bratko have just published a review of how electric fields at interfaces can modify their wettability (Top. Curr. Chem. 10.1007/128_2011_188; 2011 – paper here). Effects of this nature may play a role in the behaviour of voltage-sensitive ion channels.
Still more on denaturants. Thomas Record and colleagues at Wisconsin ask why urea is a denaturant while glycine betaine is a protein stabilizer (E. J. Guinn et al., PNAS 108, 16932-16937; 2011 – paper here). They use osmomentry and solubility measurements to look at the interactions of these molecules with 45 model proteins, and conclude that the explanation for the different behaviours lies with the details of how and where the molecules interact with the peptide surfaces. For example, urea accumulates at amide O groups, and to a lesser extent at aliphatic carbon atoms, whereas glycine betaine is excluded from them. This adds further weight to the notion that such osmolytes exert their effects via direct interactions with proteins rather than any generalized influence on ‘water structure’.
Phosphate groups turn out to be a sensitive probe of electric fields, including those that can be induced by hydration. As Steven Boxer and colleagues at Stanford show (N. M. Levinson et al., JACS 133, 13236; 2011 – paper here), electric fields perturb the vibrational spectra of organophosphates in a way that can reveal changes in hydration within partially hydrated environments, such as the active sites of enzymes.
Is there a liquid-liquid transition in confined water? That question is investigated via MD simulations by Limei Xu and Valeria Molinero at Utah (J. Phys. Chem. B jp205045k – paper here). The possibility has been raised by simulations of water in slit-like pores 2.4 nm wide (Brovchenko & Oleinikova, J. Chem. Phys. 126, 214701; 2007). Valeria and Limei use the mW water model to look at water’s behaviour in 1.5-nm hydrophilic pores at a range of temperatures and pressures up to 4000 atm. They find that at high pressures there is a signature of a somewhat abrupt but nonetheless continuous phase transition in the supercooled regime which could be interpreted as a ‘shadow’ of a L-L transition in the bulk phase which cannot itself be accessed.
A new and unusual view of the protein dynamical transition at c.200-220 K is presented in a preprint by Andrei Krokhotin and Antti Niemi at Uppsala University (paper here). They say that it this transition can be regarded as an analogue of the transition of a high-temperature superconductor to a non-superconducting pseudo-gap state. In other words, proteins can be assigned an order parameter formally equivalent to the quasiparticle wave function of superconductors. It’s not clear to me how/if this description modifies what is known already about the transition (on which, and on the role that hydration plays, there seems still to be no real consensus), but it is an original idea.
Misfolding in the context of Lum-Chandler-Weeks theory is also the subject of a paper by Ruhong Zhou and colleagues (Z. Yang et al., J. Phys. Chem. B 115, 11137 (2011) – paper here). They have looked at whether dewetting transtions play a role in the assembly of two amyloidogenic beta-sheets. For the peptides considered there are two conformations that lead to stacking of beta-sheet pairs: one containing water between the sheets in a 2D slab-like geometry, the other with the water in a 1D tube-like geometry. In both cases the sheets are brought together by a drying transition, but surprisingly this is stronger for the slab-like than the tube-like case. This is attributed here to the different surface roughness of the two packing modes: the ‘staggered’ packing in the slab-like case, which is rougher, disrupts the H-bonding network of the intervening water to a greater extent.
Some further new insights into the Lum-Chandler-Weeks dewetting mechanism for hydrophobic interactions are offered by two recent papers. Li and Walker appear to see the cross-over region between the mechanisms of hydrophobic hydration at small (<1 nm) and large (>1 nm) scales that this theory predicts, by measuring the free energy of hydration of individual monomers of various size in hydrophobic polymers, using an AFM to pull the chains out from a collapsed conformation (Li, I. T. S. & Walker, G. C. PNAS 108, 16527-16532; 2011 – paper here). At a monomer size of around 1 nm in the temperature region of 50 C or so, they find a switch in the hydration entropy from negative to positive (this crossover size reduces to just 3.5 Å at 150 C). Shekhar Garde and Amish Patel have published a commentary on it (10.1073/pnas.1113256108).
And Garde and Patel have joined forces with David Chandler and others in a preprint (arxiv.org/1109.4431 – paper here) that attempts to unravel the issue of why some protein subunits (such as melittin) seem to aggregate via dewetting while others (such as BphC) do not. They find that at model (SAM) hydrophobic surfaces, simulations show that the statistics of large-amplitude fluctuations in the density of interfacial water are altered relative to the Gaussian stats of both bulk water and water at a hydrophilic surface. In other words, despite similar average interfacial water densities, the fluctuations reveal the proximity of the hydrophobic interface to a dewetting transition. This tuning of the interfacial water is, they argue, common to biological systems, where it induces a strong sensitivity to small changes in conformation, allowing the system to take advantage of the phase transition in engineering biomolecular function (in a manner analogous to the finely balanced wetting or drying of ion channels for ‘vapour-lock’ gating). Although melittin and BphC lie on opposite sides of this transition, small modifications to both can tip the balance one way or the other. This offers what seems to me to be a persuasive argument that dewetting is relevant to hydrophobic aggregation even if it does not exactly provide the mechanism for it in all (or even in most) cases: the transition is, if you like, ‘there’ even if it doesn’t manifest itself.
Alenka Luzar and her colleagues have considered this issue from the perspective of whether or not cavitation can take place at the protein-protein interface (J. Wang et al., PCCP 10.1039/c1cp22082a – paper here). They present a lattice model which offers a fast method for predicting if cavitation can happen, and find that part of the surface of melittin is sufficiently hydrophobic to permit this on a timescale that is consistent with that seen in the earlier simulations.
Alenka and her colleagues have also examined how this putative crossover length-scale for hydration behaviour is influenced by charge on the solute (J. Stat. Phys. 10.1007/s10955-011-0337-1 – paper here). They conclude that, for moderate charge, the electrostatic contribution to the solvation free energy is in fact essentially independent of solute curvature, because of a compensation between counterion shielding and the dielectric screening of water – the solvation free energy remains more or less a function only of solute surface area.
To what extent protein-ligand binding requires an atomistic description of changes in hydration is a crucial question, not least for attempts to design synthetic ligands in drug development. Ulf Ryde at Lund University and colleagues have looked at this issue by comparing the ability of continuum methods to predict binding free energies for four different protein-ligand pairs with quite different degrees of solvent exposure at the binding site (S. Genheden et al., JACS ja202972m – paper here). They find that the continuum methods often perform badly, particularly for cases with a greater degree of solvent exposure. We need to know precisely where the waters are and where they go.
And that is somewhat elucidated by Nan-jie Deng at Rutgers University and colleagues for the case of two synthetic inhibitors of HIV-1 protease, Nelfinavir and Amprenavir (N.-j. Deng et al., J. Phys. Chem. B jp204047b – paper here). The binding of these drugs is apparently entropically driven, but the question is where that entropic contribution comes from. A classical view would be inclined to attribute it to the release of water from the binding cleft, but it seems that this isn’t so: these MD simulations suggest that any entropy gain there is more than offset by the restriction of ligand rotation and vibration on binding. Instead, the favourable entropic contribution seems to come from desolvation of the ligand.
A different view of water’s influence on bimolecular recognition is provided by Stacey Wetmore and colleagues at the University of Lethbridge in Alberta (F. M. V. Leavens et al., J. Phys. Chem. B jp205424z – paper here). They have looked at how water molecules can affect the pi-pi interactions between DNA and DNA-binding proteins. Solvating water molecules seem to have essentially no influence on the strength of pi-pi stacking for histidine and adenine, but do weaken the interaction if the histidine is protonated. The latter interaction, however, remains in all cases stronger than the former.
Protons seem to be delivered to membrane proteins such as the proton pump cytochrome c oxidase via some kind of surface-enhanced, two-dimensional transport at the membrane surface. This has been previously postulated as a series of jumps between ionisable groups (phosphate and carbonyl) at the membrane surface. But that notion is challenged by Peter Pohl at the Johannes Kepler University in Linz and colleagues, whose fluorescence measurements of proton transfer at membranes show that proton transport can be equally fast in the absence of ionisable groups (A. Springer et al., PNAS 108, 14461-14466; 2011 – paper here). They conclude that it is probably the network of interfacial water molecules that is responsible instead for the rapid proton motion: as they say, “water structuring at the interface seems to be mandatory for providing the pathway”.
It has of course been long thought that protons may be delivered to the interior of an enzyme via chains of water molecules. That process is studied by heme peroxidase by Emma Lloyd Raven and coworkers at the University of Leicester (I. Efimov et al., JACS ja2007017 – paper here). Kinetic isotope effects reveal that the proton pathway utilizes a Grotthus-like shuttling of protons along a pathway towards the ferryl oxygen that involves three bound waters and two arginine residues.
A model system for studying such proton transfer in confined geometry is reported by Bradley Habenicht and Stephen Paddison at the University of Tennessee in Knoxville (J. Phys. Chem. B jp205787f – paper here). They use MD simulations to look at how protons are transported within carbon nanotubes whose inner walls are functionalized with perfluoro sulphonic acid groups. If these groups are spaced far apart (~8 Å), they tend to be individually hydrated by clusters of water molecules with little interaction between them, and correspondingly reduced acidic proton dissociation. But there is also a pronounced effect of confinement at small nanotube diameters: in the smaller tubes there are stronger interactions between the walls and the water molecules, which can lead to break-up of the hydrogen-bonded network of waters linking the sulphonic acid groups. That network may be restored by polarized charges of fluorine atoms attached to the nanotube walls.
ATP hydrolysis in the active cleft of actin plays an important role in the state of its filamentous form F-actin, affecting its rigidity and its binding of regulatory proteins. There is water in this active site, but it hasn’t previously been clear what, if anything, it does. Marissa Saunders and Greg Voth at Chicago have clarified this through MS simulations based on the crystal structure (J. Mol. Biol. 413, 279-291; 2011 – paper here). They say that the ordered waters help the protein to flatten and brings about a conformational change that promotes ATP hydrolysis. These changes also stabilize the charge on the phosphate and accelerate the deprotonation of the catalytic water involved in hydrolysis. In short, the bound water helps to organize the active-site geometry.
Another nail in the coffin of ‘structure-making and –breaking’: Fabio Bruni and colleagues in Rome have looked at how local solvent structure around ions affects their influence on viscosity – specifically, on how changes in ionic concentration affect the viscosity of the solution (T. Corridoni et al., J. Phys. Chem. B jp202755u – paper here). Classically, the viscosity is found to be (almost) linearly related to the concentration. It has been asserted that the magnitude of the coefficient of proportionality, denoted B, depends on how the ions perturb the water structure. Fabio et al. use neutron scattering and simulations to look for some structural parameter that can be correlated with B. They find that the nature of the (univalent) ions is all but irrelevant to the size of the percolating water clusters in solution. The change in viscosity seems to be unrelated to any structural changes in the bulk liquid, but instead pertain to changes in the local hydration shells of the ions. As a result, they say, “the particular effect of solutes ranked in the Hofmeister series must be looked at in terms of specific ion interactions with hydrophilic or hydrophobic surfaces”, and not in terms of any generalized propensity for structure-making or –breaking.
In an intriguing preprint, Jampa Maruthi Pradeep Kanth and Ramesh Anishetty at the Institute of Mathematical Sciences in Chennai propose that the hydrophobic interaction should be understood as an effect analogous to the Casimir effect (the attraction of two surfaces separated by a vacuum due to the suppression of long-wavelength electromagnetic fluctuations of the vacuum in the gap) (preprint here). Their molecular mean-field analytical method suggests that confinement alters the allowed fluctuations of the hydrogen-bond network, specifically the long-ranged correlations between water molecular orientations. Now what I’d like to know is whether an explicit connection can be made to the alleged role of fluctuations in the Lum-Chandler-Weeks model. But that seems to argue in the opposite direction, namely that fluctuations are actually enhanced in the gap owing to the destabilizing influence of the hydrophobic surfaces on the intervening water layer. It’s not clear to me whether in Kanth and Anishetty the water in the gap is, aside from the suppression of fluctuations, any different from bulk water, except perhaps for the monolayer adjacent to the surfaces…? And why would this not work for hydrophilic confinement too?
Alenka Luzar and her colleagues Christopher Daub and Dusan Bratko have just published a review of how electric fields at interfaces can modify their wettability (Top. Curr. Chem. 10.1007/128_2011_188; 2011 – paper here). Effects of this nature may play a role in the behaviour of voltage-sensitive ion channels.
Still more on denaturants. Thomas Record and colleagues at Wisconsin ask why urea is a denaturant while glycine betaine is a protein stabilizer (E. J. Guinn et al., PNAS 108, 16932-16937; 2011 – paper here). They use osmomentry and solubility measurements to look at the interactions of these molecules with 45 model proteins, and conclude that the explanation for the different behaviours lies with the details of how and where the molecules interact with the peptide surfaces. For example, urea accumulates at amide O groups, and to a lesser extent at aliphatic carbon atoms, whereas glycine betaine is excluded from them. This adds further weight to the notion that such osmolytes exert their effects via direct interactions with proteins rather than any generalized influence on ‘water structure’.
Phosphate groups turn out to be a sensitive probe of electric fields, including those that can be induced by hydration. As Steven Boxer and colleagues at Stanford show (N. M. Levinson et al., JACS 133, 13236; 2011 – paper here), electric fields perturb the vibrational spectra of organophosphates in a way that can reveal changes in hydration within partially hydrated environments, such as the active sites of enzymes.
Is there a liquid-liquid transition in confined water? That question is investigated via MD simulations by Limei Xu and Valeria Molinero at Utah (J. Phys. Chem. B jp205045k – paper here). The possibility has been raised by simulations of water in slit-like pores 2.4 nm wide (Brovchenko & Oleinikova, J. Chem. Phys. 126, 214701; 2007). Valeria and Limei use the mW water model to look at water’s behaviour in 1.5-nm hydrophilic pores at a range of temperatures and pressures up to 4000 atm. They find that at high pressures there is a signature of a somewhat abrupt but nonetheless continuous phase transition in the supercooled regime which could be interpreted as a ‘shadow’ of a L-L transition in the bulk phase which cannot itself be accessed.
A new and unusual view of the protein dynamical transition at c.200-220 K is presented in a preprint by Andrei Krokhotin and Antti Niemi at Uppsala University (paper here). They say that it this transition can be regarded as an analogue of the transition of a high-temperature superconductor to a non-superconducting pseudo-gap state. In other words, proteins can be assigned an order parameter formally equivalent to the quasiparticle wave function of superconductors. It’s not clear to me how/if this description modifies what is known already about the transition (on which, and on the role that hydration plays, there seems still to be no real consensus), but it is an original idea.
Wednesday, August 17, 2011
How hydration forces assemble protein aggregates
Dielectric spectroscopy has been widely used to study the dynamics of proteins and their hydration shells, but is somewhat hampered by ambiguities about the origin of the atomistic motions that contribute to the signals. Sheila Khodadadi at NIST and colleagues have now shown just how fraught a business this is. The dielectric response of hydrated proteins typically contains two components – a fast (20-50 ps) relaxation, thought to be due to hydration water (which is slowed down relative to the bulk), and a slow (0.5-10 ns) relaxation ascribed to ‘tightly bound water’. Khodadadi and colleagues have previously questioned the former assignment (J. Phys. Chem. B 112, 14273; 2008), and now they question the latter too (J. Phys. Chem. B 115, 6222; 2011 – paper here). They say that comparisons of MD simulations to dielectric spectroscopic data suggest that the nanosecond relaxation is in fact due to motions of the protein atoms themselves, while the hydration water relaxes much more quickly.
Cytochrome c oxidase (CcO) is one of the classic examples of an enzyme that uses embedded water wires for proton transport. But how does it avoid back transport of protons along the same route? One proposal is that the water wire becomes reoriented to prevent this. Another is that a side-chain element of the hydrogen-bonded network, Glu-286, rotates to break the chain. Shuo Yang and Qiang Cui of the University of Wisconsin now show that neither process seems to be a viable gating mechanism (Biophys. J. 101, 61; 2011 – paper here). They suspect that the vectorial proton transport is probably achieved instead in a more subtle way, by stabilization of the proton-transfer transition state by the charge distribution around the active site, to explore which will require sophisticated quantum-chemical modelling.
Bacteriorhodopsin is of course another archetype for the functional water wire. It has been claimed by Klaus Gerwert at Bochum and others that the proton pumped through the protein by light absorption is stored in a water cluster inside the channel. This idea is challenged in another paper by Cui and coworkers, soon to appear in JACS (P. Goyal et al., JACS ja201568s – paper here). They say that for the proton to be stored on water requires an implausibly high increase in the pKa of a hydronium, and argue that the stored proton is instead shared between Glu194 and Glu204. One can be sure this is not going to be the last word on the matter…
I missed an interesting paper last year by Ariel Fernández and colleagues on how proteins can ‘seal’ backbone hydrogen bonds (BHBs) at their surface against competition from hydration water by a close control of the local curvature: if the BHBs are in a sufficiently highly curved location, water cannot penetrate without compromising its own H-bonded network for purely geometric reasons (E. Schultz et al., PLoS ONE 5, e12844; 2010 – paper here). Misfolded proteins fail adequately to protect their BHBs this way. This work is rather closely related to the recent paper by Fernández and Michael Lynch at Indiana on how BHBs that are ‘poorly wrapped’ by hydrophobic groups can become sites of protein-protein aggregation, which lead to complexification of the ‘interactome’ due to the degradation of wrapping created by genetic random drift (Nature 474, 502; 2011 - paper here). I have discussed that work in the August issue of Chemistry World (here – requires a subscription).
Somewhat relevant to this work is a study of protein ‘hot spots’ where preferential binding of organic small-molecule probes bind, by Frank Guarnieri of Virginia Commonwealth University in Richmond and colleagues (J. L. Kulp III et al., JACS 133, 10740; 2011 – paper here). They use simulated annealing to look at the interactions of eight probe molecules, and water, with hen egg lysozyme, and find that all the probes bind in the known binding site, which is evidently the ‘hot spot’. This implies that ligand binding isn’t simply a case of molecular recognition – the ligand is already guided towards that site by the fact that other potential binding spots are ‘guarded’ by tightly bound water. The researchers say that these hot spots can therefore also act as sites for potential protein-protein binding.
Coming back to the previous paper, Ariel also believes that poor wrapping of BHBs is implicated in the aggregation of proteins in amyloid diseases – and that these are thus potentially an alarming result of evolution’s failure to deal with random drift in anything more than an ad hoc manner. The question of how hydration is related to amyloid aggregation is also considered in a preprint by Dave Thirumalai and colleagues (arxiv 1107.4820 – paper here). They argue that “water controls the self-assembly of higher-order structures”, just as the expulsion of water is a key stage in the interaction between peptides forming beta-sheet protofilaments. Specifically, water accelerates the formation of fibrils from mainly hydrophobic peptides, but slows down the aggregation of hydrophobic sequences by stabilizing them. In the higher-order hydrophobic structures, trapped water can be considered to be analogous to water confined within carbon nanotubes – in which it seems that there is a sensitive dependence of the water structure on the width of the confining region. Thus the work nicely connects studies of confined water to an important biological problem.
A different picture – I’ve not quite figured out if it competes with or complements the above picture – of self-assembly of protein fibrils is presented by Krishnakumar Ravikumar and Wonmuk Hwang at Texas A&M (JACS 133, 11766; 2011 – paper here). They compare the role of hydration forces in the self-assembly of hydrated, dry nonpolar and dry polar surfaces, represented by collagen and two amyloidic peptides, the former a ‘steric zipper’ with interdigitating hydrophobic side-chain groups. In all cases the surfaces have hydration shells, and an oscillating hydration force as the surfaces come together due to coalescence and depletion of the hydration shells. What differs is the magnitude of these. Thus the interactions have a common origin, being water-mediated in all cases. I can’t improve on what the authors say here about the role of the hydration shells: “Thus, designating a protein surface as either “hydrophobic” or “hydrophilic” may be too simplistic of a dichotomy, as surfaces in reality lie between these idealized limits. There should be no fundamental difference in the way hydration forces arise among different types of protein surfaces, even with varying affinity for water.”
The idea that water can support two liquid states in the high-pressure supercooled regime has not been directly verified experimentally, but has received enough indirect support from both experiments and simulations to have gained wide acceptance. So a preprint from David Limmer and David Chandler at Berkeley making strong claims for the non-existence of the two liquid states is bound to cause waves (arxiv 1107.0337 – paper here). On the basis of calculations using Valeria Molinero’s recently reported mW model of water, they assert that the signatures of a phase transition seen by others relate to a solid-liquid transition, or artifacts due to imperfect equilibration (which is extremely slow in this region for, e.g. ST2 water), and that there is no sign of two liquid basins anywhere in this part of the free-energy landscape. This work, and the topic in general, will be debated in a water mini-session at the Mini Stat Mech meeting at Berkeley in January (see here), which promises to be lively.
Majed Chergui at EPFL in Lausanne and colleagues have described a nice study of the time-resolved ultrafast changes in hydration of an iodide ion as it is transformed to neutral iodine by electron abstraction, probed using XAS (V.-T. Pham et al., JACS ja203882y – paper here). This transformation changes the solute from hydrophilic to hydrophobic, and so entails a considerable reorganization of the hydration shell. The hydrogens are reoriented from pointing towards the solute to pointing away from it, forming a hydrogen-bonded ‘hydrophobic cavity’. This reorganization process takes about 3-4 ps.
Achim Müller at Bielefeld and colleagues have developed a new way to study hydrophobic species encapsulated in genuinely hydrophobic cavities. They make hollow capsules from molybdenum-based polyoxometalates, with hydrophobic interior surfaces due to ligands coordinated there (C. Schäffer et al., Chem. Eur. J. 10.1002/chem.201101454 – paper here). Much less water is found inside these shells than in those with hydrophilic interior surfaces. And hydrophobic entities such as n-hexanol are spontaneously taken up into the porous shells.
Gaurav Chopra and Michael Levitt at Stanford have used state-of-the-art quantum chemical methods to map out the hydration shell of C60 (PNAS 10.1073/pnas.1110626108 – paper here). These reveal dramatic ordering of the surrounding water, evident in the time-averaged azimuthal distribution function, which is not seen when empirical force fields are used for the calculation. Moreover, ordering of the water molecules is evident as far out as about 1 nm from the C60’s centre (as I recall, the molecule is itself about 4 Å in radius). Significantly, the azimuthal water ordering around the buckyball is not due to some kind of ice-like clathration of a non-polar species but to strong dispersion attractions between the solute and solvent molecules themselves. I guess one thing this study illustrates is the sensitivity of the important details of the hydration structure of hydrophobes to the interaction potentials used.
I think I somehow failed previously to comment on an important and provocative paper by Roy Daniel and colleagues published towards the end of last year (M. Lopez et al., Biophys. J. 99, L62; 2010 – paper here). They report significant catalytic activity of pig liver esterase in near-anhydrous conditions – not merely in a non-aqueous medium, but in a ‘dry’ powder with just 3±2 water molecules per enzyme molecule. Let me quote from the abstract: “This indicates that neither hydration water nor fast anharmonic dynamics are required for catalysis by this enzyme, implying that one of the biological requirements of water may lie with its role as a diffusion medium rather than any of its more specific properties.” They admit, naturally, that it remains an open question whether this result can be generalized to all enzymes – this one might just happen to be particularly rigid. All the same, this is most definitely a finding to chew on.
Jeremy England’s paper on allostery and hydrophobic burial, mentioned in an earlier post, has now been published in Structure (paper here). To save you a click, here’s what I said before:
The paper shows that it is possible to estimate low-energy conformational changes in a protein, such as those involved in allosteric effects, on the basis simply of residue-by-residue hydrophobic effects. Specifically, he develops a method for determining the most energetically favourable way of burying hydrophobic residues, given a particular amino-acid sequence. This amounts to identifying the particular ‘burial modes’ of any given sequence. Thus, although the stabilities of conformations are doubtless multifactorial, hydrophobicity seems to be the major governing factor.
Cytochrome c oxidase (CcO) is one of the classic examples of an enzyme that uses embedded water wires for proton transport. But how does it avoid back transport of protons along the same route? One proposal is that the water wire becomes reoriented to prevent this. Another is that a side-chain element of the hydrogen-bonded network, Glu-286, rotates to break the chain. Shuo Yang and Qiang Cui of the University of Wisconsin now show that neither process seems to be a viable gating mechanism (Biophys. J. 101, 61; 2011 – paper here). They suspect that the vectorial proton transport is probably achieved instead in a more subtle way, by stabilization of the proton-transfer transition state by the charge distribution around the active site, to explore which will require sophisticated quantum-chemical modelling.
Bacteriorhodopsin is of course another archetype for the functional water wire. It has been claimed by Klaus Gerwert at Bochum and others that the proton pumped through the protein by light absorption is stored in a water cluster inside the channel. This idea is challenged in another paper by Cui and coworkers, soon to appear in JACS (P. Goyal et al., JACS ja201568s – paper here). They say that for the proton to be stored on water requires an implausibly high increase in the pKa of a hydronium, and argue that the stored proton is instead shared between Glu194 and Glu204. One can be sure this is not going to be the last word on the matter…
I missed an interesting paper last year by Ariel Fernández and colleagues on how proteins can ‘seal’ backbone hydrogen bonds (BHBs) at their surface against competition from hydration water by a close control of the local curvature: if the BHBs are in a sufficiently highly curved location, water cannot penetrate without compromising its own H-bonded network for purely geometric reasons (E. Schultz et al., PLoS ONE 5, e12844; 2010 – paper here). Misfolded proteins fail adequately to protect their BHBs this way. This work is rather closely related to the recent paper by Fernández and Michael Lynch at Indiana on how BHBs that are ‘poorly wrapped’ by hydrophobic groups can become sites of protein-protein aggregation, which lead to complexification of the ‘interactome’ due to the degradation of wrapping created by genetic random drift (Nature 474, 502; 2011 - paper here). I have discussed that work in the August issue of Chemistry World (here – requires a subscription).
Somewhat relevant to this work is a study of protein ‘hot spots’ where preferential binding of organic small-molecule probes bind, by Frank Guarnieri of Virginia Commonwealth University in Richmond and colleagues (J. L. Kulp III et al., JACS 133, 10740; 2011 – paper here). They use simulated annealing to look at the interactions of eight probe molecules, and water, with hen egg lysozyme, and find that all the probes bind in the known binding site, which is evidently the ‘hot spot’. This implies that ligand binding isn’t simply a case of molecular recognition – the ligand is already guided towards that site by the fact that other potential binding spots are ‘guarded’ by tightly bound water. The researchers say that these hot spots can therefore also act as sites for potential protein-protein binding.
Coming back to the previous paper, Ariel also believes that poor wrapping of BHBs is implicated in the aggregation of proteins in amyloid diseases – and that these are thus potentially an alarming result of evolution’s failure to deal with random drift in anything more than an ad hoc manner. The question of how hydration is related to amyloid aggregation is also considered in a preprint by Dave Thirumalai and colleagues (arxiv 1107.4820 – paper here). They argue that “water controls the self-assembly of higher-order structures”, just as the expulsion of water is a key stage in the interaction between peptides forming beta-sheet protofilaments. Specifically, water accelerates the formation of fibrils from mainly hydrophobic peptides, but slows down the aggregation of hydrophobic sequences by stabilizing them. In the higher-order hydrophobic structures, trapped water can be considered to be analogous to water confined within carbon nanotubes – in which it seems that there is a sensitive dependence of the water structure on the width of the confining region. Thus the work nicely connects studies of confined water to an important biological problem.
A different picture – I’ve not quite figured out if it competes with or complements the above picture – of self-assembly of protein fibrils is presented by Krishnakumar Ravikumar and Wonmuk Hwang at Texas A&M (JACS 133, 11766; 2011 – paper here). They compare the role of hydration forces in the self-assembly of hydrated, dry nonpolar and dry polar surfaces, represented by collagen and two amyloidic peptides, the former a ‘steric zipper’ with interdigitating hydrophobic side-chain groups. In all cases the surfaces have hydration shells, and an oscillating hydration force as the surfaces come together due to coalescence and depletion of the hydration shells. What differs is the magnitude of these. Thus the interactions have a common origin, being water-mediated in all cases. I can’t improve on what the authors say here about the role of the hydration shells: “Thus, designating a protein surface as either “hydrophobic” or “hydrophilic” may be too simplistic of a dichotomy, as surfaces in reality lie between these idealized limits. There should be no fundamental difference in the way hydration forces arise among different types of protein surfaces, even with varying affinity for water.”
The idea that water can support two liquid states in the high-pressure supercooled regime has not been directly verified experimentally, but has received enough indirect support from both experiments and simulations to have gained wide acceptance. So a preprint from David Limmer and David Chandler at Berkeley making strong claims for the non-existence of the two liquid states is bound to cause waves (arxiv 1107.0337 – paper here). On the basis of calculations using Valeria Molinero’s recently reported mW model of water, they assert that the signatures of a phase transition seen by others relate to a solid-liquid transition, or artifacts due to imperfect equilibration (which is extremely slow in this region for, e.g. ST2 water), and that there is no sign of two liquid basins anywhere in this part of the free-energy landscape. This work, and the topic in general, will be debated in a water mini-session at the Mini Stat Mech meeting at Berkeley in January (see here), which promises to be lively.
Majed Chergui at EPFL in Lausanne and colleagues have described a nice study of the time-resolved ultrafast changes in hydration of an iodide ion as it is transformed to neutral iodine by electron abstraction, probed using XAS (V.-T. Pham et al., JACS ja203882y – paper here). This transformation changes the solute from hydrophilic to hydrophobic, and so entails a considerable reorganization of the hydration shell. The hydrogens are reoriented from pointing towards the solute to pointing away from it, forming a hydrogen-bonded ‘hydrophobic cavity’. This reorganization process takes about 3-4 ps.
Achim Müller at Bielefeld and colleagues have developed a new way to study hydrophobic species encapsulated in genuinely hydrophobic cavities. They make hollow capsules from molybdenum-based polyoxometalates, with hydrophobic interior surfaces due to ligands coordinated there (C. Schäffer et al., Chem. Eur. J. 10.1002/chem.201101454 – paper here). Much less water is found inside these shells than in those with hydrophilic interior surfaces. And hydrophobic entities such as n-hexanol are spontaneously taken up into the porous shells.
Gaurav Chopra and Michael Levitt at Stanford have used state-of-the-art quantum chemical methods to map out the hydration shell of C60 (PNAS 10.1073/pnas.1110626108 – paper here). These reveal dramatic ordering of the surrounding water, evident in the time-averaged azimuthal distribution function, which is not seen when empirical force fields are used for the calculation. Moreover, ordering of the water molecules is evident as far out as about 1 nm from the C60’s centre (as I recall, the molecule is itself about 4 Å in radius). Significantly, the azimuthal water ordering around the buckyball is not due to some kind of ice-like clathration of a non-polar species but to strong dispersion attractions between the solute and solvent molecules themselves. I guess one thing this study illustrates is the sensitivity of the important details of the hydration structure of hydrophobes to the interaction potentials used.
I think I somehow failed previously to comment on an important and provocative paper by Roy Daniel and colleagues published towards the end of last year (M. Lopez et al., Biophys. J. 99, L62; 2010 – paper here). They report significant catalytic activity of pig liver esterase in near-anhydrous conditions – not merely in a non-aqueous medium, but in a ‘dry’ powder with just 3±2 water molecules per enzyme molecule. Let me quote from the abstract: “This indicates that neither hydration water nor fast anharmonic dynamics are required for catalysis by this enzyme, implying that one of the biological requirements of water may lie with its role as a diffusion medium rather than any of its more specific properties.” They admit, naturally, that it remains an open question whether this result can be generalized to all enzymes – this one might just happen to be particularly rigid. All the same, this is most definitely a finding to chew on.
Jeremy England’s paper on allostery and hydrophobic burial, mentioned in an earlier post, has now been published in Structure (paper here). To save you a click, here’s what I said before:
The paper shows that it is possible to estimate low-energy conformational changes in a protein, such as those involved in allosteric effects, on the basis simply of residue-by-residue hydrophobic effects. Specifically, he develops a method for determining the most energetically favourable way of burying hydrophobic residues, given a particular amino-acid sequence. This amounts to identifying the particular ‘burial modes’ of any given sequence. Thus, although the stabilities of conformations are doubtless multifactorial, hydrophobicity seems to be the major governing factor.
Thursday, July 7, 2011
Stabilizing and destabilizing proteins
How do osmolytes stabilize proteins against denaturation? Using mechanically induced unfolding by means of atomic-force microscopy of protein I27 (in fact 8 repeat units in a polyprotein), Julio Fernández and colleagues at Chicago find that osmolytes can act as ‘solvent bridges’ during the unfolding transition, pinning together hydrogen-bonding sites on the polypeptide backbone (L. Dougan et al., PNAS 108, 9759; 2011 – paper here). They found in earlier studies that water molecules generally act in this capacity. They now investigate this process in the presence of glycerol, which is known to enhance protein stability, and related solutes. They find that while glycerol, ethylene glycol and propylene glycol all enhance the mechanical stability, larger hydrogen-bondong osmolytes such as sucrose and sorbitol do so to a far lesser extent – MD simulations suggest that the latter are unable to penetrate the folded structure and act as stabilizing bridges, and any stabilizing influence they exert must be indirect.
The effects of methanol on protein conformational stability are even more complex, to judge from the NMR and MD results of Christian Hilty of Texas A&M and coworkers (S. Hwang et al., J. Phys. Chem. B 115, 6653; 2011 – paper here). They report that the alcohol stabilizes the secondary structure by strengthening hydrogen-bonding interactions in the backbone (a consequence of partially eliminating the water molecules that compete with these interactions), while also weakening hydrophobic interactions and thus swelling and loosening the folded structure overall. So whether methanol tightens or loosens the structure overall is a complex balance that depends on the sequence and initial structure of the protein.
Urea and guanidinium both help to denature the model protein the Trp-cage, according to the experiments and simulations of Pavel Jungwirth in Prague and colleagues (J. Heyda et al., J. Phys. Chem. B 10.1021/jp200790h – paper here). Although these two small molecules are chemically quite different, it appears that their denaturing mechanisms here are essentially the same, involving first the destabilizing displacement (positional exchange) of two proline residues within the hydrophobic core, followed by a gradual unravelling of secondary structural elements.
The conformational stability of proteins is also influenced by ions: Hofmeister ‘salting out’ agents such as the strongly solvated, high-charge carbonate and sulphate ions also stabilize the folded state, whereas weakly solvated low-charge ions such as bromide and iodide promote denaturation. This tendency overlaps with the classical denaturation properties of complex ions such as guanidinium (Gdm+). Christopher Dempsey at Bristol and colleagues have studied these effects for the cases of the sulphates and chlorides of guanidinium and tetrapropylammonium (TPA+) (C. Dempsey et al., JACS 133, 7300; 2011 – paper here). They find that the question of conformational stabilization is subtle. TPA+, like Gdm+, perturbs the stability of some proteins (specifically the tryptophan zipper trizip, a beta-hairpin peptide), but stabilizes the alpha-helices of alahel peptides, apparently because TPA+ cannot compete effectively for hydrogen bonds in H-bond-stabilized conformations. Moreover, the cation effects may be modified by the anion: sulphate counteracts the denaturing effects of Gdm+ on trizip, but has no effect on the influence of TPA+ in that case, because Gdm+ but not TPA+ forms ion pairs with sulphate. The emerging picture is thus one in which the Hofmeister-like effects of ions must be understood in the light of a detailed consideration of (i) ion hydration; (ii) anion-cation interactions; and (iii) direct ion-protein interactions.
More on Hofmeister from Corinne Gibb and Bruce Gibb of the University of New Orleans, who offer a new perspective: namely, that so-called chaotropic anions in fact display bind preferentially to concave hydrophobic surfaces, thus effectively weakening hydrophobic attraction and promoting ‘salting in’ (JACS 133, 7344; 2011 – paper here). They reach this conclusion by looking at the thermodynamics of ion effects in the binding of adamantane carboxylic acid within the deep hydrophobic cavity of a cavitand – an interaction perturbed by anions binding to the cavity. Sure, the extension to proteins is purely by analogy, but it’s an intriguing take on the old problem. Meanwhile, Oleg Krasilnikov and colleague at the Federal University of Pernambuco in Brazil have considered how ions affect molecular interactions in nanopores, and find that these too seem to display a Hofmeister-like sequence of activities (C. G. Rodrigues et al., Biophys. J. 100, 2929; 2011 – paper here). They look at how simple ions alter the rate constant for the interaction of poly(ethylene glycol) with the protein pore alpha-hemolysin, and see a change consistent with the Hofmeister series for halide anions. They suggest that this results from a competition for hydration water between the ions and other solutes within the pore.
Protein aggregation is commonly suppressed by the addition of surfactants and sugars or polyols. But arginine hydrochloride has also been found to possess this capability, and unlike some conventional aggregation-suppressors it does so by reducing protein-protein interactions without seeming to affect the stability of the folded conformation. This behaviour hasn’t been fully explained, although Bernhardt Trout and colleagues at MIT have proposed that at least part of the mechanism might be non-specific entropic effects due to the exclusion of arginine from the gap between two proteins as they come together (B. M. Baynes et al., Biochemistry 44, 4919; 2005). They now refine this picture by looking at the influence of the anionic counterion, showing that these display the usual Hofmeister progression for aggregation suppression (C. P. Schneider et al., J. Phys. Chem. B 10.1021/jp111920y – paper here). The protonated arginine contains a guanidinium group, again potentially making the connection to denaturant activity – but the relevant behaviour here seems in fact to be the ability of the arginine ions to self-associate in stacks. Thus, while arginine can, like Gdm+, bind directly to the protein surface, the self-association weakens this interaction so that it does not cause denaturation yet still weakens protein-protein interactions. Schneider et al. elucidate this process further by looking, both experimentally and computationally, at the anion effects. Some anions, such as sulphate, phosphate and citrate, can enhance the cationic clustering by forming multiple hydrogen bonds, creating larger clusters and thus stronger exclusion effects on aggregation – and perhaps also slowing protein diffusion via an enhancement of solvent viscosity.
Understanding how bacteria void toxic substances from the cell interior could have a profound impact on our ability to combat antibiotic resistance. E. coli have a multi-drug efflux pump called AcrAB-TolC, in which the AcrB protein in the inner membrane binds drugs non-specifically and pumps them to the TolC exit duct. Attilio Vargiu of the University of Cagliari and colleagues have taken a close look at how this pump works, with a particular focus on the role of water molecules in carrying the extruded substrate along (R. Schulz et al., J. Phys. Chem. B 10.1021/jp200996x – paper here). The protein has several small holes that allow water molecules to enter and flow in a directional manner. This water acts as a lubricant and transport medium for the drug, but also flattens out the electrostatic profile in the channel that might otherwise cause the drugs to get stuck (perhaps by hydrogen bonding), and thus it contributes to the polyspecificity of the mechanism.
The role of water-mediated interactions in protein-substrate binding and associated drug design has been given a fair bit of attention, but W. David Wilson of Georgia State University and colleagues show that such things may be relevant to small-molecule DNA-binding agents too. They look at the binding of the synthetic molecule DB921 into the AT-rich minor groove of DNA, an interaction that might be useful for the disruption of parasite mitochondria (Y. Liu et al., JACS 10.1021/ja202006u – paper here). The binding is mediated by a water molecule, and to better understand how this works the researchers look at the effect on the structure, kinetics and thermodynamics of binding of introducing a host of modifications to DB291. This information, in particular the characteristics responsible for the water-mediated interaction, could be valuable for designing new agents that bind strongly in the minor groove in a sequence-specific manner.
Combining vibrational sum-frequency measurements with simulations containing three-body terms, James Skinner and colleagues at the University of Wisconsin say that the liquid-vapour interface of water shows no evidence of ‘enhanced’ molecular structuring such as ice-like ordering (P. A. Pieniazak et al., JACS 10.1021/ja2026695 – paper here).
The charge and pH of this interface have recently become contentious issues. Sylvie Roke at the MPI for Metals Research in Stuttgart and colleagues now offer a profile of the oil-water interface, using zeta-potential and sum-frequency scattering measurements alongside MD simulations (R. Vácha et al., JACS 10.1021/ja202081x – paper here). They say that the water orientations at the surface are like those at a negatively charged surface, even though there is no hydroxide absorption to make it so. There is nonetheless a surface charge, which comes instead from a disturbance in the balance of hydrogen-bond donors and acceptors at the interface.
The notion that the hydration water of proteins is dynamically coupled to the protein itself receives more support from a study by Nguyen Quang Vinh and colleagues at UCSB, who have used terahertz spectroscopy to look at the large-scale collective vibrations of lysozyme (N. Q. Vinh et al., JACS 133, 8942; 2011 – paper here). They find that the protein is surrounded by 150-180 water molecules (a sub-monolayer) that, in the authors’ words, ‘in terms of their picosecond dynamics behave as if they are an integral part of the protein’. THz spectroscopy seems to be emerging as a nigh-incomparable technique for probing these long-ranged collective motions.
The dynamics of hydration water are also studied by Cesare Cametti at the University of Rome ‘La Sapienza’ and colleagues, using dielectic spectroscopy (C. Cametti et al., J. Phys. Chem. B 10.1021/jp2019389 – paper here). They find that the dielectric relaxation of the lysozyme hydration sphere is bimodal at high concentrations, corresponding to tightly and loosely bound waters, although monomodal in dilute solution.
The nature of the protein dynamical transition at 200-220 K shows no sign of being resolved. Salvatore Magazù and colleagues at the University of Messina now throw a cat among the pigeons by suggesting that there is no such transition at all: it is an artefact caused by the coincidence of the system’s relaxation time with the instrumental resolution (S. Magazù et al., J. Phys. Chem. B 115, 7736; 2011 – paper here). Nonetheless, they say – can life really be this complicated? – there is a crossover of some sort at 220 K, at least for the case of lysozyme considered here, for this marks a change from Arrhenius to super-Arrhenius behaviour in the coupled hydration-water/protein motions. I think what this implies – it is a little unclear – is that there is no intrinsic, qualitative change in the protein dynamics at the ‘dynamical transition’, but rather, a change due to the coupling of these to the hydration water dynamics. I could be wrong.
Water rotational dynamics are slowed down around many small solutes, both hydrophilic and hydrophobic. So it stands to reason that the same should happen for larger solutes, such as proteins. And it does – but Ana Vila Verde and R. Kramer Campen at the FOM Institute in Amsterdam suggest that the latter is not necessarily just a straightforward extension of the former (J. Phys. Chem. B 10.1021/jp112178c – paper here). Their simulations of water dynamics around the disaccharides kojibiose and trehalose show that the sheer size of these solutes, relative to smaller ones, induces additional mechanisms of water retardation as a result of topological constraints on water motions. Thus, one can’t for example imagine that the hydration of a free amino acid is the same as that when the amino acid represents a peptide residue.
Bear that in mind, perhaps, in considering the hydration structure of glycine as deduced from ab initio calculations by Bo Liu at Henan University and colleagues. They build up the hydration shell molecule by molecule from a gas-phase picture (Y. Yao et al., J. Phys. Chem. B 115, 6213; 2011 – paper here).
Salt bridges play a role in stabilizing the glycosyl hydrolase (an enzyme with potentially important industrial applications) of the hyperthermophile Rhodothermus marinus (L. Bleicher et al., J. Phys. Chem. B 115, 7940; 2011 – paper here). But perhaps surprisingly, the enzyme also contains salt bridges that seem to be destabilizing: located in the hydrophobic core, where they might facilitate the permeation of water.
C. Preston Moon and Karen Fleming at Johns Hopkins have a neat idea for developing a hydrophobicity scale for amino acids based on their energy of transfer from water to within a phospholipid bilayer, thus relating the measure directly to the driving forces for the assembly and stabilization of membrane proteins (PNAS 108, 10174; 2011 – paper here). Their thermodynamics measurements show some differences from the predictions of simulations, especially for the translocation of arginines – which makes the results relevant to voltage-sensitive ion channel gating mechanisms, since these involve the movement of arginines into the hydrophobic interior of the membrane.
How homogeneous are concentrated aqueous solutions? This question has been studied for methanol, which seems to aggregate to some extent in water; now Lorna Dougan at the University of Leeds and colleagues study the case of glycerol using neutron scattering, motivated by the relevance to cryoprotection (J. J. Towey et al., J. Phys. Chem. B 115, 7799; 2011 – paper here). They find that glycerol-glycerol hydrogen-bonding in the pure liquid is scarcely affected by the addition of water, while the water-water bonding is highly disrupted. In effect, the waters become isolated from one another, binding preferentially to glycerols. Thus, it seems likely that glycerol could act as a cryoprotectant by keeping water molecules apart and preventing for the formation of an ice network.
Water can penetrate the hydrophobic interior of carbon nanotubes, a fact that is being investigated for potential desalination technologies among other things. William Goddard at Caltech and colleagues take a look at what drives the filling process for different tube diameters (T. A. Pascal et al., PNAS pnas.1108073108 – paper here). For CNTs between 0.8 and 2.7 nm, the interior water phase is always more stable than the bulk, but for several different reasons. For nanotubes thinner than 1 nm, the water phase is gas-like, with an entropic driving force. For nanotubes of 1.1-1.2 nm the encapsulated phase is ice-like and enthalpy-stabilized. For nanotubes wider than 1.4 nm the interior phase is liquid-like, but stabilized by increased translational energy. The overall message is sobering in showing how very fine adjustments to the hydrogen-bonded network and the balance of interactions with the surrounding environment can create quite different phase behaviour and thermodynamic driving forces in confined situations even for very small differences in dimensions.
A new method for rapidly calculating solvation energies in water is presented by Jianzhong Wu and coworkers at the University of California at Riverside (S. Zhao et al., J. Phys. Chem. B 10.1021/jp201949k – paper here), which combines DFT with MD simulations. The researchers have so far tested it for simple ions, for which it works well. Meanwhile, Merchant and Dilip Asthagiri at JHU have a preprint (arxiv 1106.0448 – paper here) in which they examine the range of ion-specific effects in water and conclude that, at least for sodium, potassium, chloride and fluoride, these extend no more than about 4 Å, so not much more than the size of a single water molecule.
This hasn’t yet exhausted my list of papers: still to come (soon) are developments on the putative liquid-liquid transition of water and on proton transport in bacteriorhodopsin…
The effects of methanol on protein conformational stability are even more complex, to judge from the NMR and MD results of Christian Hilty of Texas A&M and coworkers (S. Hwang et al., J. Phys. Chem. B 115, 6653; 2011 – paper here). They report that the alcohol stabilizes the secondary structure by strengthening hydrogen-bonding interactions in the backbone (a consequence of partially eliminating the water molecules that compete with these interactions), while also weakening hydrophobic interactions and thus swelling and loosening the folded structure overall. So whether methanol tightens or loosens the structure overall is a complex balance that depends on the sequence and initial structure of the protein.
Urea and guanidinium both help to denature the model protein the Trp-cage, according to the experiments and simulations of Pavel Jungwirth in Prague and colleagues (J. Heyda et al., J. Phys. Chem. B 10.1021/jp200790h – paper here). Although these two small molecules are chemically quite different, it appears that their denaturing mechanisms here are essentially the same, involving first the destabilizing displacement (positional exchange) of two proline residues within the hydrophobic core, followed by a gradual unravelling of secondary structural elements.
The conformational stability of proteins is also influenced by ions: Hofmeister ‘salting out’ agents such as the strongly solvated, high-charge carbonate and sulphate ions also stabilize the folded state, whereas weakly solvated low-charge ions such as bromide and iodide promote denaturation. This tendency overlaps with the classical denaturation properties of complex ions such as guanidinium (Gdm+). Christopher Dempsey at Bristol and colleagues have studied these effects for the cases of the sulphates and chlorides of guanidinium and tetrapropylammonium (TPA+) (C. Dempsey et al., JACS 133, 7300; 2011 – paper here). They find that the question of conformational stabilization is subtle. TPA+, like Gdm+, perturbs the stability of some proteins (specifically the tryptophan zipper trizip, a beta-hairpin peptide), but stabilizes the alpha-helices of alahel peptides, apparently because TPA+ cannot compete effectively for hydrogen bonds in H-bond-stabilized conformations. Moreover, the cation effects may be modified by the anion: sulphate counteracts the denaturing effects of Gdm+ on trizip, but has no effect on the influence of TPA+ in that case, because Gdm+ but not TPA+ forms ion pairs with sulphate. The emerging picture is thus one in which the Hofmeister-like effects of ions must be understood in the light of a detailed consideration of (i) ion hydration; (ii) anion-cation interactions; and (iii) direct ion-protein interactions.
More on Hofmeister from Corinne Gibb and Bruce Gibb of the University of New Orleans, who offer a new perspective: namely, that so-called chaotropic anions in fact display bind preferentially to concave hydrophobic surfaces, thus effectively weakening hydrophobic attraction and promoting ‘salting in’ (JACS 133, 7344; 2011 – paper here). They reach this conclusion by looking at the thermodynamics of ion effects in the binding of adamantane carboxylic acid within the deep hydrophobic cavity of a cavitand – an interaction perturbed by anions binding to the cavity. Sure, the extension to proteins is purely by analogy, but it’s an intriguing take on the old problem. Meanwhile, Oleg Krasilnikov and colleague at the Federal University of Pernambuco in Brazil have considered how ions affect molecular interactions in nanopores, and find that these too seem to display a Hofmeister-like sequence of activities (C. G. Rodrigues et al., Biophys. J. 100, 2929; 2011 – paper here). They look at how simple ions alter the rate constant for the interaction of poly(ethylene glycol) with the protein pore alpha-hemolysin, and see a change consistent with the Hofmeister series for halide anions. They suggest that this results from a competition for hydration water between the ions and other solutes within the pore.
Protein aggregation is commonly suppressed by the addition of surfactants and sugars or polyols. But arginine hydrochloride has also been found to possess this capability, and unlike some conventional aggregation-suppressors it does so by reducing protein-protein interactions without seeming to affect the stability of the folded conformation. This behaviour hasn’t been fully explained, although Bernhardt Trout and colleagues at MIT have proposed that at least part of the mechanism might be non-specific entropic effects due to the exclusion of arginine from the gap between two proteins as they come together (B. M. Baynes et al., Biochemistry 44, 4919; 2005). They now refine this picture by looking at the influence of the anionic counterion, showing that these display the usual Hofmeister progression for aggregation suppression (C. P. Schneider et al., J. Phys. Chem. B 10.1021/jp111920y – paper here). The protonated arginine contains a guanidinium group, again potentially making the connection to denaturant activity – but the relevant behaviour here seems in fact to be the ability of the arginine ions to self-associate in stacks. Thus, while arginine can, like Gdm+, bind directly to the protein surface, the self-association weakens this interaction so that it does not cause denaturation yet still weakens protein-protein interactions. Schneider et al. elucidate this process further by looking, both experimentally and computationally, at the anion effects. Some anions, such as sulphate, phosphate and citrate, can enhance the cationic clustering by forming multiple hydrogen bonds, creating larger clusters and thus stronger exclusion effects on aggregation – and perhaps also slowing protein diffusion via an enhancement of solvent viscosity.
Understanding how bacteria void toxic substances from the cell interior could have a profound impact on our ability to combat antibiotic resistance. E. coli have a multi-drug efflux pump called AcrAB-TolC, in which the AcrB protein in the inner membrane binds drugs non-specifically and pumps them to the TolC exit duct. Attilio Vargiu of the University of Cagliari and colleagues have taken a close look at how this pump works, with a particular focus on the role of water molecules in carrying the extruded substrate along (R. Schulz et al., J. Phys. Chem. B 10.1021/jp200996x – paper here). The protein has several small holes that allow water molecules to enter and flow in a directional manner. This water acts as a lubricant and transport medium for the drug, but also flattens out the electrostatic profile in the channel that might otherwise cause the drugs to get stuck (perhaps by hydrogen bonding), and thus it contributes to the polyspecificity of the mechanism.
The role of water-mediated interactions in protein-substrate binding and associated drug design has been given a fair bit of attention, but W. David Wilson of Georgia State University and colleagues show that such things may be relevant to small-molecule DNA-binding agents too. They look at the binding of the synthetic molecule DB921 into the AT-rich minor groove of DNA, an interaction that might be useful for the disruption of parasite mitochondria (Y. Liu et al., JACS 10.1021/ja202006u – paper here). The binding is mediated by a water molecule, and to better understand how this works the researchers look at the effect on the structure, kinetics and thermodynamics of binding of introducing a host of modifications to DB291. This information, in particular the characteristics responsible for the water-mediated interaction, could be valuable for designing new agents that bind strongly in the minor groove in a sequence-specific manner.
Combining vibrational sum-frequency measurements with simulations containing three-body terms, James Skinner and colleagues at the University of Wisconsin say that the liquid-vapour interface of water shows no evidence of ‘enhanced’ molecular structuring such as ice-like ordering (P. A. Pieniazak et al., JACS 10.1021/ja2026695 – paper here).
The charge and pH of this interface have recently become contentious issues. Sylvie Roke at the MPI for Metals Research in Stuttgart and colleagues now offer a profile of the oil-water interface, using zeta-potential and sum-frequency scattering measurements alongside MD simulations (R. Vácha et al., JACS 10.1021/ja202081x – paper here). They say that the water orientations at the surface are like those at a negatively charged surface, even though there is no hydroxide absorption to make it so. There is nonetheless a surface charge, which comes instead from a disturbance in the balance of hydrogen-bond donors and acceptors at the interface.
The notion that the hydration water of proteins is dynamically coupled to the protein itself receives more support from a study by Nguyen Quang Vinh and colleagues at UCSB, who have used terahertz spectroscopy to look at the large-scale collective vibrations of lysozyme (N. Q. Vinh et al., JACS 133, 8942; 2011 – paper here). They find that the protein is surrounded by 150-180 water molecules (a sub-monolayer) that, in the authors’ words, ‘in terms of their picosecond dynamics behave as if they are an integral part of the protein’. THz spectroscopy seems to be emerging as a nigh-incomparable technique for probing these long-ranged collective motions.
The dynamics of hydration water are also studied by Cesare Cametti at the University of Rome ‘La Sapienza’ and colleagues, using dielectic spectroscopy (C. Cametti et al., J. Phys. Chem. B 10.1021/jp2019389 – paper here). They find that the dielectric relaxation of the lysozyme hydration sphere is bimodal at high concentrations, corresponding to tightly and loosely bound waters, although monomodal in dilute solution.
The nature of the protein dynamical transition at 200-220 K shows no sign of being resolved. Salvatore Magazù and colleagues at the University of Messina now throw a cat among the pigeons by suggesting that there is no such transition at all: it is an artefact caused by the coincidence of the system’s relaxation time with the instrumental resolution (S. Magazù et al., J. Phys. Chem. B 115, 7736; 2011 – paper here). Nonetheless, they say – can life really be this complicated? – there is a crossover of some sort at 220 K, at least for the case of lysozyme considered here, for this marks a change from Arrhenius to super-Arrhenius behaviour in the coupled hydration-water/protein motions. I think what this implies – it is a little unclear – is that there is no intrinsic, qualitative change in the protein dynamics at the ‘dynamical transition’, but rather, a change due to the coupling of these to the hydration water dynamics. I could be wrong.
Water rotational dynamics are slowed down around many small solutes, both hydrophilic and hydrophobic. So it stands to reason that the same should happen for larger solutes, such as proteins. And it does – but Ana Vila Verde and R. Kramer Campen at the FOM Institute in Amsterdam suggest that the latter is not necessarily just a straightforward extension of the former (J. Phys. Chem. B 10.1021/jp112178c – paper here). Their simulations of water dynamics around the disaccharides kojibiose and trehalose show that the sheer size of these solutes, relative to smaller ones, induces additional mechanisms of water retardation as a result of topological constraints on water motions. Thus, one can’t for example imagine that the hydration of a free amino acid is the same as that when the amino acid represents a peptide residue.
Bear that in mind, perhaps, in considering the hydration structure of glycine as deduced from ab initio calculations by Bo Liu at Henan University and colleagues. They build up the hydration shell molecule by molecule from a gas-phase picture (Y. Yao et al., J. Phys. Chem. B 115, 6213; 2011 – paper here).
Salt bridges play a role in stabilizing the glycosyl hydrolase (an enzyme with potentially important industrial applications) of the hyperthermophile Rhodothermus marinus (L. Bleicher et al., J. Phys. Chem. B 115, 7940; 2011 – paper here). But perhaps surprisingly, the enzyme also contains salt bridges that seem to be destabilizing: located in the hydrophobic core, where they might facilitate the permeation of water.
C. Preston Moon and Karen Fleming at Johns Hopkins have a neat idea for developing a hydrophobicity scale for amino acids based on their energy of transfer from water to within a phospholipid bilayer, thus relating the measure directly to the driving forces for the assembly and stabilization of membrane proteins (PNAS 108, 10174; 2011 – paper here). Their thermodynamics measurements show some differences from the predictions of simulations, especially for the translocation of arginines – which makes the results relevant to voltage-sensitive ion channel gating mechanisms, since these involve the movement of arginines into the hydrophobic interior of the membrane.
How homogeneous are concentrated aqueous solutions? This question has been studied for methanol, which seems to aggregate to some extent in water; now Lorna Dougan at the University of Leeds and colleagues study the case of glycerol using neutron scattering, motivated by the relevance to cryoprotection (J. J. Towey et al., J. Phys. Chem. B 115, 7799; 2011 – paper here). They find that glycerol-glycerol hydrogen-bonding in the pure liquid is scarcely affected by the addition of water, while the water-water bonding is highly disrupted. In effect, the waters become isolated from one another, binding preferentially to glycerols. Thus, it seems likely that glycerol could act as a cryoprotectant by keeping water molecules apart and preventing for the formation of an ice network.
Water can penetrate the hydrophobic interior of carbon nanotubes, a fact that is being investigated for potential desalination technologies among other things. William Goddard at Caltech and colleagues take a look at what drives the filling process for different tube diameters (T. A. Pascal et al., PNAS pnas.1108073108 – paper here). For CNTs between 0.8 and 2.7 nm, the interior water phase is always more stable than the bulk, but for several different reasons. For nanotubes thinner than 1 nm, the water phase is gas-like, with an entropic driving force. For nanotubes of 1.1-1.2 nm the encapsulated phase is ice-like and enthalpy-stabilized. For nanotubes wider than 1.4 nm the interior phase is liquid-like, but stabilized by increased translational energy. The overall message is sobering in showing how very fine adjustments to the hydrogen-bonded network and the balance of interactions with the surrounding environment can create quite different phase behaviour and thermodynamic driving forces in confined situations even for very small differences in dimensions.
A new method for rapidly calculating solvation energies in water is presented by Jianzhong Wu and coworkers at the University of California at Riverside (S. Zhao et al., J. Phys. Chem. B 10.1021/jp201949k – paper here), which combines DFT with MD simulations. The researchers have so far tested it for simple ions, for which it works well. Meanwhile, Merchant and Dilip Asthagiri at JHU have a preprint (arxiv 1106.0448 – paper here) in which they examine the range of ion-specific effects in water and conclude that, at least for sodium, potassium, chloride and fluoride, these extend no more than about 4 Å, so not much more than the size of a single water molecule.
This hasn’t yet exhausted my list of papers: still to come (soon) are developments on the putative liquid-liquid transition of water and on proton transport in bacteriorhodopsin…
Tuesday, May 24, 2011
Hydration of PSII
What a beautiful crystal structure is reported by Yasufumi Umena of Osaka City University and colleagues in Nature (473, 55; 2011 – paper here). They have revealed the entire photosystem II complex at 1.9 Å resolution, showing the hydration structure of the core Mn4CaO5 cluster along with the locations of 1,300 water molecules in a hydration shell that seems to offer several hydrogen-bonded channels for protons, water molecules for photolysis, or oxygen molecules. The latter are probably formed by oxidation of some of the four waters bound to the Mn cluster. I don’t recall ever seeing before such a complex, orchestrated and carefully rationalized example of a multifunctional hydration structure.
Collagen is generally considered a structural protein, but particular collagen motifs are also recognized as substrates by collagen binding proteins such as adhesins in some human pathogens. On the basis of MD simulations and comparison with crystal structures, Luigi Vitagliano at the Consiglio Nazionale delle Ricerce in Naples and colleagues say that the regions of collagen-binding adhesin CNA involved in binding to hydrophobic parts of the collagen triple helix are inherently prone to dewetting, making them primed to bind their target by reducing the desolvation penalty (Vitagliano et al., Biophys. J. 100, 2253-2261; 2011 – paper here). Moreover, besides these hydrophobic contacts the interaction between CNA and collagen is mediated by an intricate network of 13 water molecules.
Local water densities around biological systems can be calculated fairly accurately with a computationally cheap interaction-site model (based on the Ornstein-Zernicke integral equation), rather than with a full MD simulation, according to a new study by Vijay Pande at Stanford and colleagues, who compare the two for the hydration of GroEL (M. C. Stumpe et al., J. Phys. Chem. B 115, 319; 2011 – paper here).
In drug design, the surfaces of target proteins are often mapped out to look for ‘hot spots’ where it is particularly advantageous to place functional groups in the ligand complementary to those on the protein. This process often identifies many such local minima in a rugged potential-energy surface. But that’s in vacuo for a rigid surface. One might expect that including the protein’s conformational flexibility and interactions with water will smooth out this rugged landscape. But Katrina Lexa and Heather Carlson at the University of Michigan say that it doesn’t, unless the protein is allowed its full flexibility (JACS 133, 200; 2011 – paper here). In other words, there are no short cuts: if the molecule is semi-rigid, spurious hot spots remain.
Structured water molecules bound within cytochrome c have been suspected for some time of participating in the enzyme’s electron-transfer and oxygen reduction reactions, but it hasn’t been clear exactly how. Amandine Maréchal and Peter Rich at University College London have used FTIR spectroscopy to investigate the issue (PNAS pnas.1019419108 – paper here). They find that rearrangements of up to 8 water molecules are associated with the photolysis reaction, probably forming transient hydrogen-bonded pathways for proton conduction and gating.
More roles for water in protein-ligand binding are revealed by Michelle Sahai and Philip Biggin at Oxford (J. Phys. Chem. B 10.1021/jp200776t – paper here). They consider how the GluA2 ionotropic glutamate receptor binds both glutamate and the related compound AMPA, and find two quite distinct modes of binding. The difference seems to be in the location of a single water molecule in the binding cleft. They use density functional theory to figure out why it is more favourable for the water to sit in different positions in the two different cases. That they don’t seem yet to have fully settled that question (they cannot consider entropic contributions) seems to underline how tricky it might be to use bound waters in rational drug design.
Joe Dzubiella in Berlin has an interesting preprint on the thermodynamics of hydrophobic association which claims that the curvature of the interface is important. He points out how there seems to be a crossover at small size scales (about 1 nm) between enthalpy-driven hydrophobic association at larger scales and entropy-driven at smaller scales. But for concave binding cavities this does not seem to apply: enthalpy continues to dominate even at very small scales. Joe explains this on the basis that the surface-area-based models used to describe large-scale interactions on the basis of solvent-accessible area and surface tension remain applicable at small scales so long as “the antagonistic effects on concave vs. convex bending on water interface thermodynamics are properly taken into account”. The paper will appear in a forthcoming special issue of J. Stat. Phys. dedicated to water.
There’s a nice potted summary of current understanding of antifreeze protein ice-binding mechanisms by Kim Sharp of the University of Pennsylvania in PNAS (pnas.1104618108 – paper here). It is a commentary on a new study by Garnham et al. (PNAS pnas.1100429108), which I haven’t yet got hold of. They report the crystal structure of the AFP of an Antarctic bacterium, Marinomonas primoryensis, which has a new binding motif, a parallel beta-helix. This structure has the ice-binding surface fully solvent-exposed in the crystalline state, and so is likely to be “free from crystal-packing artifacts.” Much of this surface is hydrophobic (although anchored at the edges by H-bonds), and the claim is that this induces a clathrate-like structure in the first hydration layer that is close to the structure of ice – in other words, the AFP “brings its own ‘ice’ with it”.
With an eye on the Lum/Chandler/Weeks model of hydrophobic attraction, Pablo Debenedetti and colleagues have calculated the evaporation length scale – the separation of solvophobic plates at which capillary evaporation occurs – for water and a range of organic liquids (C. A. Cerdeiriña et al., J. Phys. Chem. Lett. 2, 1000; 2011 – paper here). There’s nothing conceptually new here, but the numbers are somewhat surprising: for water they find a length scale of about 1.5 microns at atmospheric pressure, which is much larger than I’d have expected. However, this applies for purely repulsive surfaces (contact angle of 180 degrees), which is of course rarely found, and never for protein surfaces. The length is also large for the organics, such as benzene, heptane and cyclohexane, but about a factor of 3 less so – water is (somewhat) anomalous here because of its large surface tension.
Hydration seems to respond to the flexibility of alicyclic systems, according to Annalisa Boscaino and Kevin Naidoo of the University of Cape Town in South Africa (J. Phys. Chem. B 10.1021/jp110248j – paper here). They find from MD simulations that molecules with a cyclopyranose framework, such as glucose, have a significantly higher hydration number than those based on a cyclohexane framework with hydroxyls, such as cyclohexanol. This seems to result from the greater rigidity of the former, enabling the formation of longer-lived hydrogen bonds to the surrounding water.
It’s generally thought that rearrangements of the hydrogen-bond network in bulk water must have a collective character. Andrei Tokmakoff at MIT and colleagues now provide evidence of that (R. A. Nicodemus et al., J. Phys. Chem. B 115, 5604; 2011 – paper here). They have used ultrafast IR spectroscopy of HOD in pure water to measure the energy barriers to spectral diffusion and reorientational relaxation, and find that the slow-decay component is consistent with collective reorganization.
Marcus Weinwurm and Christoph Dellago have calculated the vibrational spectra of single-file water molecules in narrow pores, enabling them to distinguish it from the stacked-ring structure in wider pores (J. Phys. Chem. B 115, 5268; 2011 – paper here).
Collagen is generally considered a structural protein, but particular collagen motifs are also recognized as substrates by collagen binding proteins such as adhesins in some human pathogens. On the basis of MD simulations and comparison with crystal structures, Luigi Vitagliano at the Consiglio Nazionale delle Ricerce in Naples and colleagues say that the regions of collagen-binding adhesin CNA involved in binding to hydrophobic parts of the collagen triple helix are inherently prone to dewetting, making them primed to bind their target by reducing the desolvation penalty (Vitagliano et al., Biophys. J. 100, 2253-2261; 2011 – paper here). Moreover, besides these hydrophobic contacts the interaction between CNA and collagen is mediated by an intricate network of 13 water molecules.
Local water densities around biological systems can be calculated fairly accurately with a computationally cheap interaction-site model (based on the Ornstein-Zernicke integral equation), rather than with a full MD simulation, according to a new study by Vijay Pande at Stanford and colleagues, who compare the two for the hydration of GroEL (M. C. Stumpe et al., J. Phys. Chem. B 115, 319; 2011 – paper here).
In drug design, the surfaces of target proteins are often mapped out to look for ‘hot spots’ where it is particularly advantageous to place functional groups in the ligand complementary to those on the protein. This process often identifies many such local minima in a rugged potential-energy surface. But that’s in vacuo for a rigid surface. One might expect that including the protein’s conformational flexibility and interactions with water will smooth out this rugged landscape. But Katrina Lexa and Heather Carlson at the University of Michigan say that it doesn’t, unless the protein is allowed its full flexibility (JACS 133, 200; 2011 – paper here). In other words, there are no short cuts: if the molecule is semi-rigid, spurious hot spots remain.
Structured water molecules bound within cytochrome c have been suspected for some time of participating in the enzyme’s electron-transfer and oxygen reduction reactions, but it hasn’t been clear exactly how. Amandine Maréchal and Peter Rich at University College London have used FTIR spectroscopy to investigate the issue (PNAS pnas.1019419108 – paper here). They find that rearrangements of up to 8 water molecules are associated with the photolysis reaction, probably forming transient hydrogen-bonded pathways for proton conduction and gating.
More roles for water in protein-ligand binding are revealed by Michelle Sahai and Philip Biggin at Oxford (J. Phys. Chem. B 10.1021/jp200776t – paper here). They consider how the GluA2 ionotropic glutamate receptor binds both glutamate and the related compound AMPA, and find two quite distinct modes of binding. The difference seems to be in the location of a single water molecule in the binding cleft. They use density functional theory to figure out why it is more favourable for the water to sit in different positions in the two different cases. That they don’t seem yet to have fully settled that question (they cannot consider entropic contributions) seems to underline how tricky it might be to use bound waters in rational drug design.
Joe Dzubiella in Berlin has an interesting preprint on the thermodynamics of hydrophobic association which claims that the curvature of the interface is important. He points out how there seems to be a crossover at small size scales (about 1 nm) between enthalpy-driven hydrophobic association at larger scales and entropy-driven at smaller scales. But for concave binding cavities this does not seem to apply: enthalpy continues to dominate even at very small scales. Joe explains this on the basis that the surface-area-based models used to describe large-scale interactions on the basis of solvent-accessible area and surface tension remain applicable at small scales so long as “the antagonistic effects on concave vs. convex bending on water interface thermodynamics are properly taken into account”. The paper will appear in a forthcoming special issue of J. Stat. Phys. dedicated to water.
There’s a nice potted summary of current understanding of antifreeze protein ice-binding mechanisms by Kim Sharp of the University of Pennsylvania in PNAS (pnas.1104618108 – paper here). It is a commentary on a new study by Garnham et al. (PNAS pnas.1100429108), which I haven’t yet got hold of. They report the crystal structure of the AFP of an Antarctic bacterium, Marinomonas primoryensis, which has a new binding motif, a parallel beta-helix. This structure has the ice-binding surface fully solvent-exposed in the crystalline state, and so is likely to be “free from crystal-packing artifacts.” Much of this surface is hydrophobic (although anchored at the edges by H-bonds), and the claim is that this induces a clathrate-like structure in the first hydration layer that is close to the structure of ice – in other words, the AFP “brings its own ‘ice’ with it”.
With an eye on the Lum/Chandler/Weeks model of hydrophobic attraction, Pablo Debenedetti and colleagues have calculated the evaporation length scale – the separation of solvophobic plates at which capillary evaporation occurs – for water and a range of organic liquids (C. A. Cerdeiriña et al., J. Phys. Chem. Lett. 2, 1000; 2011 – paper here). There’s nothing conceptually new here, but the numbers are somewhat surprising: for water they find a length scale of about 1.5 microns at atmospheric pressure, which is much larger than I’d have expected. However, this applies for purely repulsive surfaces (contact angle of 180 degrees), which is of course rarely found, and never for protein surfaces. The length is also large for the organics, such as benzene, heptane and cyclohexane, but about a factor of 3 less so – water is (somewhat) anomalous here because of its large surface tension.
Hydration seems to respond to the flexibility of alicyclic systems, according to Annalisa Boscaino and Kevin Naidoo of the University of Cape Town in South Africa (J. Phys. Chem. B 10.1021/jp110248j – paper here). They find from MD simulations that molecules with a cyclopyranose framework, such as glucose, have a significantly higher hydration number than those based on a cyclohexane framework with hydroxyls, such as cyclohexanol. This seems to result from the greater rigidity of the former, enabling the formation of longer-lived hydrogen bonds to the surrounding water.
It’s generally thought that rearrangements of the hydrogen-bond network in bulk water must have a collective character. Andrei Tokmakoff at MIT and colleagues now provide evidence of that (R. A. Nicodemus et al., J. Phys. Chem. B 115, 5604; 2011 – paper here). They have used ultrafast IR spectroscopy of HOD in pure water to measure the energy barriers to spectral diffusion and reorientational relaxation, and find that the slow-decay component is consistent with collective reorganization.
Marcus Weinwurm and Christoph Dellago have calculated the vibrational spectra of single-file water molecules in narrow pores, enabling them to distinguish it from the stacked-ring structure in wider pores (J. Phys. Chem. B 115, 5268; 2011 – paper here).
Thursday, April 28, 2011
Water as glue
There is a very nice crop of papers to draw on for this post, many of which speak to very central questions in this field. First up is a paper in Nature Communications from Volkhard Helms and colleagues at Saarbrücken, which looks at the detailed role of interfacial water in the association of hydrophilic protein surfaces (M. Ahmad et al., Nat. Commun. 2, 261; 2011 – paper here). Whereas hydrophobic association has been the focus of a lot of recent attention, especially with a view to the possibility of a dewetting-induced process, hydrophilic surfaces have received less attention. That’s an important lacuna, since as the authors point out, around 70% of interfacial residues are hydrophilic. The common approach is to assume direct electrostatic interaction mediated by a continuum solvent. But the water network has a more complex role. As shown in these MD simulations of the barnase-barstar complex, water molecules mediate and stabilize the interactions between native contacts. Moreover, for electrostatic interactions to be important, the interfacial water’s dielectric constant needs to be reduced to reduce screening. This happens as a consequence of changes in the structure of the interfacial layers (the dielectric permittivity is less than 50% of the bulk value for interfacial separations of less than 1.2 nm), and it preferentially promotes electrostatic interactions normal to the surfaces. In other words, you could say that (once again, though arguably to put the cart before the horse) water does exactly what is required of it.
Jeremy England, now at Princeton, has a paper in press with Structure which shows that it is possible to estimate low-energy conformational changes in a protein, such as those involved in allosteric effects, on the basis simply of residue-by-residue hydrophobic effects. Specifically, he develops a method for determining the most energetically favourable way of burying hydrophobic residues, given a particular amino-acid sequence. This amounts to identifying the particular ‘burial modes’ of any given sequence. Thus, although the stabilities of conformations are doubtless multifactorial, hydrophobicity seems to be the major governing factor.
Calculating the surface free energies of heterogeneous surfaces exposed to water (such as protein surfaces) is tough. The approach of the Cassie equation is additive, but as Alenka Luzar and colleagues point out, that doesn’t always work (J. Wang et al., PNAS 108, 6374-6379; 2011 – paper here). They show that deviations from linear additivity result when parts of a surface are unevenly exposed to solvent. In particular, it seems that polar patches exert an inordinately strong influence, being able to ‘pin’ a droplet so that it might remain closely attached to adjacent hydrophobic patches. They examine these effects with reference to water droplets first on a functionalized graphene surface and then on the surface of melittin.
Daryl Eggers at San José State University presents an interesting approach to the energetics of reactions in aqueous solution, geared especially to biochemical equilibria, that treats the water as a reactant and product, thereby subsuming the local changes in water structure that inevitably accompany the reaction (Biochemistry 50, 2004-2012; 2011 – paper here). Here the free energy of bulk water is treated as a variable, allowing for the effects of all solutes including those that may not participate directly in the reaction (such as dissolved salts). As I understand it, this offers a means of accommodating the effects of such solutes (for example, in salting in/out) that does not make any assumptions about global changes in ‘water structure’, but represents only the global average of localized changes. Also jettisoned in that process is any insistence on putative structure-makers and structure-breakers; rather, solvation effects need be discussed only in what seems like relatively uncontentious terms of subpopulations of water with differing free energies. I haven’t yet quite figured out how one gets at these free energies in experimental terms, but I like the principle, not least because it explicitly acknowledges the role of water as a participant.
Yingkai Zhang at New York University and coworkers have used ab initio MD simulations to study the mechanism of action of histone deacetylase (HDAC) enzymes, which remove acetyl groups from histone residues and have been identified as a target for anti-cancer drugs (R. Wu et al., JACS 133, 6110-6113; 2011 – paper here). They suggest that some HDACs function via a mechanism involving a modulation of water access (a hydrogen-bonded chain of waters) to the binding pocket, in which a zinc ion in the metalloenzyme binds to its substrate. The presence or absence of water alters the dielectric constant in the binding pocket and thereby affects the strength of zinc binding.
Transitions of DNA between A, B and Z forms are thought to be associated with and perhaps driven by transitions in the nature of hydration. Karim Fahmy and colleagues at the Institute of Radiochemistry in Dresden now suggest that the same applies to the more subtle sub-transitions between the BI and BII states of the B-form (H. Khesbak et al., JACS 133, 5834-5842; 2011 – paper here). Specifically, there are two sub-populations of water molecules – one bound to phosphates, the other not – that contribute to stabilizing the two conformations via entropic effects. These water rearrangements can also be involved in interactions with DNA-binding ligands, such as the antimicrobial peptide indolicidin, by a water-mediated induced fit.
The structures of amyloid fibrillar assemblies are still far from well understood. The hydration state of the peptides is a particularly important issue in determining their stability and perhaps their mode of formation. Beat Meier at ETH and colleagues report some tricks that enable this question to be probed by NMR (Van Melckebeke et al., J. Mol. Biol. 405, 765; 2011 – paper here). For a particular prion domain called HET-s(218-289) they show that, although these protofibrils have a hydrophobic core and a semi-hydrophobic pocket, they do not engage in ‘dry’ interfibril contacts but are each surrounded by water.
The behaviour of water close to and between lipid bilayers has been much studied, but there doesn’t seem to have been much consideration of how that behaviour might feature in biological membrane processes such as fusion. This issue is investigated by Vijay Pande at Stanford and colleagues using simulations (P. M. Kasson, E. Lindahl & V. S. Pande, JACS 133, 3812-3815; 2011 – paper here). They look at the water trapped between the faces of two approaching membranes – that is, in hydrophilic confinement – and find that the dynamics are altered significantly. Specifically, the trapped water has reduced rotational entropy, and it helps the two membranes to adhere. However, the slower dynamics also retards the process of fusion itself – the formation of the ‘stalk’ that bridges the lipid membranes.
Mafumi Hishida and Koichiro Tanaka at Kyoto have also looked at the hydration of phospholipid bilayers, here experimentally using terahertz spectroscopy and SAXS (Phys. Rev. Lett. 106, 158102; 2011 – paper here). They conclude that water structure is perturbed up to 4-5 layers from the surface, over a distance of at least 1 nm, and that the average density in this hydration layer is slightly greater than that in the bulk.
Meanwhile, Joshua Layfield and Diego Troya at Virginia Tech have considered a water droplet confined between hydrophobic surfaces of self-assembled monolayers (J. Phys. Chem. B 115, 4662-4670; 2011 – paper here). Here the water motions (lateral translational diffusion) are accelerated by confinement, and this effect seems to operate over distances of more than 1 nm from the surfaces. Structural effects (preferential orientation of the water molecules) aren’t evident beyond 1 nm, however – but while the authors consider this to be a relatively short-ranged effect, I’d have been surprised to see anything structural with a longer reach.
Daniela Russo at the ILL and colleagues have used inelastic neutron scattering to probe the low-frequency densities of states of water hydrating small ‘model peptides’ (N-acetyl-leucine-methylamide, NALMA, and N-acetyl-glycine-methylamide, NAGMA) at low temperatures (Russo et al., JACS 133, 4882-4888; 2011 – paper here). At 200K they find that the hydration water for the hydrophilic NAGMA is similar to high-density amorphous ice, while that of the hydrophobic NALMA is more like low-density amorphous ice. Something similar has been reported at 100 K by Paciaroni et al. (Phys. Rev. Lett. 101, 148104; 2008), but was in that case attributed to curvature of the biomolecular surface – which is evidently not the case for these small molecules.
Aquaporins seem to play a crucial role in water regulation in arid periods during the life cycle of the major malaria vector mosquito Anopheles gambiae, according to Kun Liu of the Johns Hopkins Malaria Research Institute in Baltimore and colleagues (K. Liu et al., PNAS 108, 6062-6066; 2011 – paper here). The authors don’t say whether this makes these AQPs a potential target for controlling the spread of malaria, though I suppose that is a possible implication.
There’s still more to be understood about what hydrogen bonds are, as evidenced by a recent IUPAC working group set up to redefine them (see here). Angelos Michaelides at UCL (who I have to thank for my water-crested football shirt) and colleagues have now refined the quantum picture of the H-bond, showing how quantum nuclear effects due to the anharmonicity of the bond and the small proton mass can alter the bond strength, weakening weak H-bonds (like those in water) and strengthening strong ones (X.-Z. Li et al., PNAS 108, 6369-6373; 2011 – paper here).
There are one or two other papers I’ve still to get to, including some that folks have kindly sent to me. Apologies for that – more soon, I hope.
Jeremy England, now at Princeton, has a paper in press with Structure which shows that it is possible to estimate low-energy conformational changes in a protein, such as those involved in allosteric effects, on the basis simply of residue-by-residue hydrophobic effects. Specifically, he develops a method for determining the most energetically favourable way of burying hydrophobic residues, given a particular amino-acid sequence. This amounts to identifying the particular ‘burial modes’ of any given sequence. Thus, although the stabilities of conformations are doubtless multifactorial, hydrophobicity seems to be the major governing factor.
Calculating the surface free energies of heterogeneous surfaces exposed to water (such as protein surfaces) is tough. The approach of the Cassie equation is additive, but as Alenka Luzar and colleagues point out, that doesn’t always work (J. Wang et al., PNAS 108, 6374-6379; 2011 – paper here). They show that deviations from linear additivity result when parts of a surface are unevenly exposed to solvent. In particular, it seems that polar patches exert an inordinately strong influence, being able to ‘pin’ a droplet so that it might remain closely attached to adjacent hydrophobic patches. They examine these effects with reference to water droplets first on a functionalized graphene surface and then on the surface of melittin.
Daryl Eggers at San José State University presents an interesting approach to the energetics of reactions in aqueous solution, geared especially to biochemical equilibria, that treats the water as a reactant and product, thereby subsuming the local changes in water structure that inevitably accompany the reaction (Biochemistry 50, 2004-2012; 2011 – paper here). Here the free energy of bulk water is treated as a variable, allowing for the effects of all solutes including those that may not participate directly in the reaction (such as dissolved salts). As I understand it, this offers a means of accommodating the effects of such solutes (for example, in salting in/out) that does not make any assumptions about global changes in ‘water structure’, but represents only the global average of localized changes. Also jettisoned in that process is any insistence on putative structure-makers and structure-breakers; rather, solvation effects need be discussed only in what seems like relatively uncontentious terms of subpopulations of water with differing free energies. I haven’t yet quite figured out how one gets at these free energies in experimental terms, but I like the principle, not least because it explicitly acknowledges the role of water as a participant.
Yingkai Zhang at New York University and coworkers have used ab initio MD simulations to study the mechanism of action of histone deacetylase (HDAC) enzymes, which remove acetyl groups from histone residues and have been identified as a target for anti-cancer drugs (R. Wu et al., JACS 133, 6110-6113; 2011 – paper here). They suggest that some HDACs function via a mechanism involving a modulation of water access (a hydrogen-bonded chain of waters) to the binding pocket, in which a zinc ion in the metalloenzyme binds to its substrate. The presence or absence of water alters the dielectric constant in the binding pocket and thereby affects the strength of zinc binding.
Transitions of DNA between A, B and Z forms are thought to be associated with and perhaps driven by transitions in the nature of hydration. Karim Fahmy and colleagues at the Institute of Radiochemistry in Dresden now suggest that the same applies to the more subtle sub-transitions between the BI and BII states of the B-form (H. Khesbak et al., JACS 133, 5834-5842; 2011 – paper here). Specifically, there are two sub-populations of water molecules – one bound to phosphates, the other not – that contribute to stabilizing the two conformations via entropic effects. These water rearrangements can also be involved in interactions with DNA-binding ligands, such as the antimicrobial peptide indolicidin, by a water-mediated induced fit.
The structures of amyloid fibrillar assemblies are still far from well understood. The hydration state of the peptides is a particularly important issue in determining their stability and perhaps their mode of formation. Beat Meier at ETH and colleagues report some tricks that enable this question to be probed by NMR (Van Melckebeke et al., J. Mol. Biol. 405, 765; 2011 – paper here). For a particular prion domain called HET-s(218-289) they show that, although these protofibrils have a hydrophobic core and a semi-hydrophobic pocket, they do not engage in ‘dry’ interfibril contacts but are each surrounded by water.
The behaviour of water close to and between lipid bilayers has been much studied, but there doesn’t seem to have been much consideration of how that behaviour might feature in biological membrane processes such as fusion. This issue is investigated by Vijay Pande at Stanford and colleagues using simulations (P. M. Kasson, E. Lindahl & V. S. Pande, JACS 133, 3812-3815; 2011 – paper here). They look at the water trapped between the faces of two approaching membranes – that is, in hydrophilic confinement – and find that the dynamics are altered significantly. Specifically, the trapped water has reduced rotational entropy, and it helps the two membranes to adhere. However, the slower dynamics also retards the process of fusion itself – the formation of the ‘stalk’ that bridges the lipid membranes.
Mafumi Hishida and Koichiro Tanaka at Kyoto have also looked at the hydration of phospholipid bilayers, here experimentally using terahertz spectroscopy and SAXS (Phys. Rev. Lett. 106, 158102; 2011 – paper here). They conclude that water structure is perturbed up to 4-5 layers from the surface, over a distance of at least 1 nm, and that the average density in this hydration layer is slightly greater than that in the bulk.
Meanwhile, Joshua Layfield and Diego Troya at Virginia Tech have considered a water droplet confined between hydrophobic surfaces of self-assembled monolayers (J. Phys. Chem. B 115, 4662-4670; 2011 – paper here). Here the water motions (lateral translational diffusion) are accelerated by confinement, and this effect seems to operate over distances of more than 1 nm from the surfaces. Structural effects (preferential orientation of the water molecules) aren’t evident beyond 1 nm, however – but while the authors consider this to be a relatively short-ranged effect, I’d have been surprised to see anything structural with a longer reach.
Daniela Russo at the ILL and colleagues have used inelastic neutron scattering to probe the low-frequency densities of states of water hydrating small ‘model peptides’ (N-acetyl-leucine-methylamide, NALMA, and N-acetyl-glycine-methylamide, NAGMA) at low temperatures (Russo et al., JACS 133, 4882-4888; 2011 – paper here). At 200K they find that the hydration water for the hydrophilic NAGMA is similar to high-density amorphous ice, while that of the hydrophobic NALMA is more like low-density amorphous ice. Something similar has been reported at 100 K by Paciaroni et al. (Phys. Rev. Lett. 101, 148104; 2008), but was in that case attributed to curvature of the biomolecular surface – which is evidently not the case for these small molecules.
Aquaporins seem to play a crucial role in water regulation in arid periods during the life cycle of the major malaria vector mosquito Anopheles gambiae, according to Kun Liu of the Johns Hopkins Malaria Research Institute in Baltimore and colleagues (K. Liu et al., PNAS 108, 6062-6066; 2011 – paper here). The authors don’t say whether this makes these AQPs a potential target for controlling the spread of malaria, though I suppose that is a possible implication.
There’s still more to be understood about what hydrogen bonds are, as evidenced by a recent IUPAC working group set up to redefine them (see here). Angelos Michaelides at UCL (who I have to thank for my water-crested football shirt) and colleagues have now refined the quantum picture of the H-bond, showing how quantum nuclear effects due to the anharmonicity of the bond and the small proton mass can alter the bond strength, weakening weak H-bonds (like those in water) and strengthening strong ones (X.-Z. Li et al., PNAS 108, 6369-6373; 2011 – paper here).
There are one or two other papers I’ve still to get to, including some that folks have kindly sent to me. Apologies for that – more soon, I hope.
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