The debate rumbles on over Hofmeister effects. In a paper in Scholarly Research Exchange [doi:10.3814/2008/761829 – paper here], Terence Evens and Randall Niedz of the US Horticultural Research Laboratory in Florida say that many previous studies of ion-specific effects on protein precipitation are flawed because they fail to take into account the dependence of pH on the type and concentration of ions in solution, treating it as an independent variable. More generally, they say that individual ion effects can’t be deduced in any straightforward way from the effects of specific salts. In a nutshell, this seems to be the key message: ‘Is the sulphate ion more effective at protein precipitation than the chloride ion? It depends on the protein. It depends on protein concentration. It depends on the concentration of the respective ions. It depends on the proportions and concentrations of the other cations and anions in solution. It depends on the dissolved gases. It may or may not depend on the pH. It depends on temperature. These dependencies are conflated, confounded, lost or ignored in traditional Hofmeister series, but are fundamentally essential to realizing a deeper understanding of ion-specific effects.’ Discuss, as they say. It’s certainly a rather discouraging message on what is already a bewildering problem, but Evens and Niedz present results for ovalbumin and BSA that seem to bear out this complexity.
In a related vein, Shekhar Garde and colleagues at RPI have examined the thermodyanmcis of hydrophobic hydration, association and folding for a hydrophobic polymer in sodium chloride solution and aqueous trimethylamine oxide (TMAO), an osmolyte [M. V. Athawale et al., J. Phys. Chem B 112, 5661; 2008 – paper here]. They’ve found previously that NaCl weakens hydrophobic hydration and enhances association, while TMAO has little effect (Ghosh et al., J. Phys. Chem. B 109, 642; 2005 and Athawale et al., Biophys. J. 89, 858; 2005). Here they carry out temperature-dependent simulations to figure out if the effects are entropic or enthalpic. For TMAO, there is almost precise enthalpic-entropic compensation. For NaCl, changes in solvent-solvent, solvent-salt and salt-salt energy lead to a dominant enthalpic contribution at small length scales (that is, for small solutes), but the strengthening of hydrophobic interactions is entropic in origin at large length scales, being governed by the need to form a solvent-solute interface. This seems to offer further evidence that there is no single ‘explanation’ of Hofmeister-type effects.
Meanwhile, Agustín Colussi and colleagues at Caltech have returned to a more basic level of the problem: the fractionation of ions at the air-water interface (a loose proxy for the air-hydrophobe interface) [J. Cheng et al., J Phys. Chem. B 112, 7157; 2008 – paper here]. They have shown previously [J. Cheng et al., J. Phys. Chem. B 110, 25598; 2006] that aggregation of anions at the interface seems to increase with increasing ion radius. They now extend their experimental study to the cases of the large anion PF6- and the highly polarizable IO3-, and look also at the effect of adding methanol, which will migrate to the surface and cap it with methyl groups. The same relationship with ion radius is found, and the fractionation barely depends on the methanol content. The authors conclude that this fractionation happens not because the ions have any affinity with the surface but because they are expelled from the bulk.
Now forget the salts. Esben Thormann and colleagues at the University of Southern Denmark have looked again at a familiar model system: a polystyrene particle several microns across stuck to an AFM tip and brought close to hydrophobic and hydrophilic surfaces [E. Thormann et al., Langmuir doi:10.1021/la8005162 – paper here]. For approaching surfaces in the hydrophilic case, all looks fine: the interactions are described by DLVO theory. But for the hydrophobic case, bridging air bubbles form, as has often been hypothesized, leading to jump-in at a separation of around 10 nm due to the action of the meniscus. When the particle is retracted, the bubble becomes elongated until it ruptures at about 70 nm. In both cases there are also force plateaus at separations of up to a few hundred nm, which the researchers interpret in terms of bridging polymer molecules pulled out from the particle surface. All this argues for caution in regarding the system as a model of the biological case.
Let’s stick with these model surfaces for a bit. Some time ago I mentioned some ‘curious’ results of Andrei Sommer and colleagues at the University of Ulm on irradiation of water films on diamond. I found some difficulty there figuring out what the underlying hypothesis was. Andrei has now sent me more material on this. The basic motivation for the work is the fact, known for some time but unexplained, that the surfaces of diamond are somewhat conductive. Andrei and colleagues believe this is due to proton migration in thin surface films of water, which are formed in humid conditions. Their experiments [A. P. Sommer et al., Cryst. Growth. Design 7, 2298; 2007] show that for hydrogen-terminated diamond, the conductivity drcreases with increasing humidity. They think this is because the highly ordered water films that form at low humidity are disrupted, degrading proton motion, as the films get thicker. This idea challenges the widely accepted model for the surface conductivity, called the transfer doping model [M. I. Landstrass & K. V. Ravi, Appl. Phys. Lett. 55, 975; 1989], which would predict increased conductivity with increased humidity. Andrei and colleagues have recently debated this point with John Angus and colleagues in Science [V. Chakrapani et al., Science 318, 1424; 2007].
From the perspective of water in biology, Andrei suggests the key point is that the highly ordered (indeed, essentially crystalline) water nanofilms he identifies on the (hydrophobic) diamond surface offer “a unique platform for the systematic investigation of nanoscopic water layers.” In a forthcoming paper for Crystal Growth and Design, he and his colleagues Dan Zhu and Hans Fecht argue that these layers might even provide a platform for the origin of life, as I understand it by potentially templating the evolution of organic monolayers. Apparently Albert Szent-Györgyi suggested something similar in the 1970s, proposing such a role for crystalline interfacial water layers. Diamonds can be extremely ancient, and also extraterrestrial. All this is very intriguing, although as someone now programmed to approach with scepticism the notion of enhanced ordering of water at hydrophobic surfaces I think I would like to see some more direct evidence that the water molecules on diamond are indeed truly ordered, especially if the claim is that this extends beyond a monolayer. But I think they’re working on that.
Heme catalases convert hydrogen peroxide to water and oxygen. One type of such enzyme, so-called Clade 3 of the most abundant (monofunctional) class, contains a tightly bound NADPH molecule which seems to protect one of the intermediates of the ferryloxo group against deactivation to a catalytically inactive form. Reiner Sustmann at Duisburg-Essen and colleagues propose in a new paper (W. Sicking et al., JACS 130, 7345-7356; 2008 – paper here) that a bound water molecule plays a critical part in this process, both by supplying a hydroxyl group that binds temporarily to the porphyrin group and then assists the fast two-electron reduction of the intermediate ferryloxo species by NADPH via a series of proton shifts, to restore the catalase resting state and avoid diversion of the reaction towards the deactivated state. A nice example of the multiple, sophisticated roles that bound water can play in active sites.
Lei Zhou and Steven Siegelbaum at Columbia University present a new coarse-grained approach for conducting normal-mode analysis of the dynamics of proteins, which has a lower computational cost than trying to extract the dynamics from a full MD simulation with explicit water [Biophys. J. 94, 3461; 2008 – paper here]. They say that this method is more accurate than are existing coarse-grained NMA techniques, and gives good agreement with experimental results from quasieleastic neutron and light scattering.
In my last blog entry I referred to recent work on the excited-state dynamics of the green fluorescent protein. Dan Huppert and colleagues at Tel Aviv University have looked at essentially the same aspect of the problem: the role of the proton-transfer process [R. Gepshtein et al., Langmuir 112, 7203; 2008 – paper here]. They say that the non-exponential dynamics seem to stem from the distance-dependence of the proton transfer between the chromophore and a bound water molecule that acts as the acceptor. This distance has a relatively large spread of about 0.2 angstroms in GFP.
Finally, thanks to everyone who helped make my Chem. Rev. article a most-accessed paper for the period Jan-Mar 2008.
Thursday, June 26, 2008
Monday, June 16, 2008
A mixed bag
Michael Fayer and colleagues at Stanford have looked at how high salt concentrations and nanoconfinement alter orientational relaxation of water’s hydrogen-bonded network using ultrafast IR spectroscopy [S. Park et al., J. Phys. Chem. B 112, 5279-5290; 2008 – paper here.] They find that structural rearrangements of the network are slowed in 6M NaBr, but only moderately – by a factor around 3. The effects of confinement in reverse micelles can be more pronounced, being up to 20 times slower when the ‘nanopools’ of enclosed water are just 1.7 nm across. Moreover, the relaxation then becomes non-exponential. The effect seems to be due more to the effects of confinement per se than to interactions with the charged lipid head groups.
Jim Hynes and Damien Laage have a paper [J. Phys. Chem. B 10.1021/jp802033r] reporting an improved method for determining water residence times in hydration shells in MD simulations, which works with anything from ions to proteins. The key, it seems, is a better handling of the ‘tolerance time’, which relates to frustrated attempts of a water molecule to escape from the first hydration shell.
The spectrum of the green fluorescent protein of the Pacific jellyfish Aequorea Victoria, widely used in molecular biology as a marker, has several absorption bands that are interpreted as resulting from protonation and ionization of certain residues. The excited-state dynamics are thought to involve a proton relay involving three protons that can shuttle along a chain involving a bound water molecule. Ricard Gelabert of the Universitat Autònoma de Barcelona and colleagues have studied this process using a nuclear quantum dynamical simulation, and they find that proton transfer can be extremely fast in this system, initially happening in a matter of femtoseconds (but slowing down in the final stages). Moreover, the three protons seem to travel synchronously along the relay. The transfer induces a conformational change that breaks the relay, and thus is irreversible. The paper [O. Vendrell et al., J. Phys. Chem. B 112, 5500-5511; 2008] is here.
Nikolai Smolin and Valerie Daggett in Seattle have studied the mechanism of a so-called type III antifreeze protein from polar pout (Macrozoarces americanus) using simulations (J. Phys. Chem. B 112, 6193-6202; 2008 – paper here). They’re trying to figure out which of the various possible mechanisms for AFPs seems to apply here, and find that hydration waters on the protein’s ice-binding surface are more tetrahedral and ice-like than those elsewhere in the hydration sphere, suggesting that there is a good epitaxial match that promotes the binding of the protein to incipient ice crystals, preventing their further growth. I’d be interested to know if/how one might rule out the possibility that the protein could in fact provide a site for ice nucleation this way, preventing the growth of large crystals via a proliferation of small ones.
Sinan Keten and Markus Buehler at MIT have an interesting paper in Phys. Rev. Lett. (100, 198301; paper here) on the strength of protein folds secured purely by hydrogen-bonding. They use concepts from conventional fracture mechanics to look at the rupture of H-bonded beta-sheet-like folds, which enables them to conclude that protein domains stabilized this way can’t have rupture forces greater than about 200 pN.
A couple of papers in Langmuir look at the nature of the water-solid interface. Bill Ducker and colleagues have studied the formation of nanobubbles at hydrophobic surfaces, using total-internal-reflection IR spectroscopy to confirm that there are genuine gas-phase molecules present in both air and CO2 bubbles, some as small as just a few nm across to judge from the AFM images also presented (X. H. Zhang et al., Langmuir 24, 4756-4764; 2008 – paper here). The pressures are estimated at around 1-1.7 atm, but while the air bubbles can be stable for days, CO2 bubbles persist for only an hour or two. So while these nanobubbles are not ubiquitous on hydrophobic surfaces, they do form quite routinely.
And Sergio Acuna and Pedro Toledo in Chile have measured short-range forces between glass surfaces in water, using the AFM (Langmuir 24, 4881-4887; 2008 – paper here here). They find a repulsion at short distances (an intervening water film of 3-4 molecular layers) that does not depend on pH or on ion concentration or size. They say that the mechanism of silica hairs, proposed by Israelachvili and Wennerström (Nature 379, 219-225; 1996), can’t explain their data, and that the oscillatory forces they see are due to sequential squeezing out of water layers. I don’t fully understand what the authors mean by attributing this to the ‘creation of a hydrogen-bonding network at the surface level’ – whether this is different from the bulk, say, and why one need invoke hydrogen bonding at all as opposed simply to the kinds of steric packing effects that create oscillatory solvation forces in any solvent.
The paper on water dynamics in cells by Marion Jasnin, Joe Zaccai and colleagues that I mentioned earlier is now published in EMBO Reports, and is available here.
To those who’ve sent me material: I firmly intend to comment on it soon!
Jim Hynes and Damien Laage have a paper [J. Phys. Chem. B 10.1021/jp802033r] reporting an improved method for determining water residence times in hydration shells in MD simulations, which works with anything from ions to proteins. The key, it seems, is a better handling of the ‘tolerance time’, which relates to frustrated attempts of a water molecule to escape from the first hydration shell.
The spectrum of the green fluorescent protein of the Pacific jellyfish Aequorea Victoria, widely used in molecular biology as a marker, has several absorption bands that are interpreted as resulting from protonation and ionization of certain residues. The excited-state dynamics are thought to involve a proton relay involving three protons that can shuttle along a chain involving a bound water molecule. Ricard Gelabert of the Universitat Autònoma de Barcelona and colleagues have studied this process using a nuclear quantum dynamical simulation, and they find that proton transfer can be extremely fast in this system, initially happening in a matter of femtoseconds (but slowing down in the final stages). Moreover, the three protons seem to travel synchronously along the relay. The transfer induces a conformational change that breaks the relay, and thus is irreversible. The paper [O. Vendrell et al., J. Phys. Chem. B 112, 5500-5511; 2008] is here.
Nikolai Smolin and Valerie Daggett in Seattle have studied the mechanism of a so-called type III antifreeze protein from polar pout (Macrozoarces americanus) using simulations (J. Phys. Chem. B 112, 6193-6202; 2008 – paper here). They’re trying to figure out which of the various possible mechanisms for AFPs seems to apply here, and find that hydration waters on the protein’s ice-binding surface are more tetrahedral and ice-like than those elsewhere in the hydration sphere, suggesting that there is a good epitaxial match that promotes the binding of the protein to incipient ice crystals, preventing their further growth. I’d be interested to know if/how one might rule out the possibility that the protein could in fact provide a site for ice nucleation this way, preventing the growth of large crystals via a proliferation of small ones.
Sinan Keten and Markus Buehler at MIT have an interesting paper in Phys. Rev. Lett. (100, 198301; paper here) on the strength of protein folds secured purely by hydrogen-bonding. They use concepts from conventional fracture mechanics to look at the rupture of H-bonded beta-sheet-like folds, which enables them to conclude that protein domains stabilized this way can’t have rupture forces greater than about 200 pN.
A couple of papers in Langmuir look at the nature of the water-solid interface. Bill Ducker and colleagues have studied the formation of nanobubbles at hydrophobic surfaces, using total-internal-reflection IR spectroscopy to confirm that there are genuine gas-phase molecules present in both air and CO2 bubbles, some as small as just a few nm across to judge from the AFM images also presented (X. H. Zhang et al., Langmuir 24, 4756-4764; 2008 – paper here). The pressures are estimated at around 1-1.7 atm, but while the air bubbles can be stable for days, CO2 bubbles persist for only an hour or two. So while these nanobubbles are not ubiquitous on hydrophobic surfaces, they do form quite routinely.
And Sergio Acuna and Pedro Toledo in Chile have measured short-range forces between glass surfaces in water, using the AFM (Langmuir 24, 4881-4887; 2008 – paper here here). They find a repulsion at short distances (an intervening water film of 3-4 molecular layers) that does not depend on pH or on ion concentration or size. They say that the mechanism of silica hairs, proposed by Israelachvili and Wennerström (Nature 379, 219-225; 1996), can’t explain their data, and that the oscillatory forces they see are due to sequential squeezing out of water layers. I don’t fully understand what the authors mean by attributing this to the ‘creation of a hydrogen-bonding network at the surface level’ – whether this is different from the bulk, say, and why one need invoke hydrogen bonding at all as opposed simply to the kinds of steric packing effects that create oscillatory solvation forces in any solvent.
The paper on water dynamics in cells by Marion Jasnin, Joe Zaccai and colleagues that I mentioned earlier is now published in EMBO Reports, and is available here.
To those who’ve sent me material: I firmly intend to comment on it soon!
Wednesday, May 21, 2008
Effects of confinement
I have been feeling guilty at the slow pace with which I’ve been reading through a very nice preprint sent to me some time ago by Bob Eisenberg at Rush University Medical Center in Chicago, on the topic of how bubbles might act to gate ion channels. This brings together many recent ideas on how protein channels might be gated by evacuation of water from a hydrophobic pore – exactly the sort of thing discussed in the reviews by Gerhard Hummer et al. and Haiping Fang et al. in my previous post. The notion is that different channels have different mechanisms – mechanical, say, or voltage-depedent – to modulate the hydrophobicity of the channel and thus to cause an abrupt transition to a dewetted, capillary-evaporated state in which solutes are precluded from the channel. Eisenberg and colleagues present a general thermodynamic analysis of this process, and also offer the hypothesis that such a ‘bubble-induced’ mechanism might explain the anaesthetic effects of inert gases. The point is that the authors have now published the paper – or at least, what I assume is the same paper, as I’ve only so far seen the abstract – in Biophys. J. 94, 4282-4298 (2008) (paper here). Well worth looking at.
On the same general topic, Niharendu Choudhury in Mumbai has used MD simulations to look at how dewetting and capillary evaporation between two hydrophobic plates (close-packed paraffin monolayers) depends on the fine details of the plate structure (J. Phys. Chem. B 112, 6296-6300; 2008 – paper here). Specifically, he examines how the behaviour of the nano-confined water layer depends not only on plate separation but on the intermolecular distance in the paraffin plates, allowing a kind of ‘dilution’ of the hydrophobicity and solvent-surface dispersion forces. He finds that tuning this parameter can cause switches between wet, dry and intermittent wet/dry states, which might help to resolve differences seen in previous studies of this geometry. Moreover, the flickering formation and break-up of a water layer in the intermittent state can happen on nanosecond timescales, implying that simulations of protein hydration lasting only a few picoseconds may overlook important dynamical aspects of the problem.
Tobias Cramer at the University of Bologna and colleagues have looked at what one might consider the complementary problem: the spontaneous formation of a water bridge between two proximal surfaces (Langmuir 10.1021/la800220r – paper here). Their MD simulations examine how this process depends on an electric field across the gap, showing that there is a critical field strength at which the inhibitory influence of surface tension is overcome by electrostatic pressure. The focus here is mostly on scanning-probe-microscope-based experiments and technologies such as dip-pen nanolithography, but one can presumably imagine charging mechanisms for drawing water columns inside otherwise hydrophobic cavities in biomolecules. I have no idea whether such things are observed in nature…
There’s another take on nanoconfined water in a paper by Matthew Lane and colleagues at Sandia (J. M. D. Lane et al., Langmuir 24, 5209-5212; 2008 – paper here). They study the dynamics of a very thin film of water (submonolayer to bilayer) between two carboxyl-terminated alkanethiol self-assembled monolayers, using MD simulations. The diffusion coefficient of the water decreases as the layers become thinner, down to two orders of magnitude less than the bulk value, but the water remains liquid-like.
David Chandler, working with Adam Willard, has more on the role of solvent fluctuations in his dewetting model of hydrophobic assembly (discussed in earlier posts) in a paper in J. Phys. Chem. B 112, 6187-6192; 2008 (paper here). Fluctuations play a crucial role in the formation of a ‘vapour tunnel’ between two spherical hydrophobic particles, which draws them together. And in the same volume, Peter Rossky and colleagues expand on their work on the mechanism of protein cold denaturation, which I’ve also mentioned previously (C. F. Lopez et al., J. Phys. Chem. B 112, 5961-5967; 2008 – paper here).
Here’s another nice example of bound water playing a crucial role in enzyme function in a paper by Sason Shaik at the Hebrew University of Jerusalem and colleagues (Y. Wang et al., JACS 10.1021/ja711426y). They have looked at the mechanism by which cytochrome P450 StaP catalyses the formation of staurosporine, an antitumour agent, from chromopyrrolic acid. A critical step in this process is the abstraction of a proton from an N-H group on the substrate by an iron-oxo species in the enzyme. This seems to happen with the concerted assistance of two water molecules in the binding site: one shifts the proton onto a nearby histidine residue, and the other takes a proton from the other side of this residue’s side-chain and puts it on the iron-oxo group. Thus, the water molecules here form a hydrogen-bonded proton-relay network.
Rohit Pappu and colleagues at the University of St Louis in Missouri have an interesting study on the conformation of intrinsically disordered proteins, a class of protein that lack well defined 3D structures (H. T. Tran et al., JACS 10.1021/ja710446s – paper here). The common notion seems to be that because these proteins have sequences of low hydrophobicity, they are not tightly bound into compact structures by hydrophobic interactions. But IDPs are not totally random – they do have an ensemble of preferred conformational states. What creates them? The authors use simulations to conclude that these states are not dominated by specific intramolecular interactions in the polypeptide backbone, but from solvent-solute interactions, since water seems to be a generically poor solvent even for these low-hydrophobicity backbones.
More on hydration of poorly folded proteins comes from Supid Chakraborty and Sanjoy Bandyopadhyay at the Indian Institute of Technology in Kharagpur. They have used simulations to look at how the unfolding of the HP-36 subdomain of villin headpiece protein affects the dynamics of the hydration shell (J. Phys. Chem. B 112, 6500-6507; 2008 – paper here). Unfolding turns out to have a strong but quite complex effect on the rotational and translational motions of water in the hydration shell. It seems possible that there are knock-on effects: as one part of the protein unfolds, this can alter the structure and dynamics of hydration water around other segments in a cooperative manner. There’s clearly much more to be done on this interesting but under-investigated issue.
There’s more, as ever, to come when time permits…
On the same general topic, Niharendu Choudhury in Mumbai has used MD simulations to look at how dewetting and capillary evaporation between two hydrophobic plates (close-packed paraffin monolayers) depends on the fine details of the plate structure (J. Phys. Chem. B 112, 6296-6300; 2008 – paper here). Specifically, he examines how the behaviour of the nano-confined water layer depends not only on plate separation but on the intermolecular distance in the paraffin plates, allowing a kind of ‘dilution’ of the hydrophobicity and solvent-surface dispersion forces. He finds that tuning this parameter can cause switches between wet, dry and intermittent wet/dry states, which might help to resolve differences seen in previous studies of this geometry. Moreover, the flickering formation and break-up of a water layer in the intermittent state can happen on nanosecond timescales, implying that simulations of protein hydration lasting only a few picoseconds may overlook important dynamical aspects of the problem.
Tobias Cramer at the University of Bologna and colleagues have looked at what one might consider the complementary problem: the spontaneous formation of a water bridge between two proximal surfaces (Langmuir 10.1021/la800220r – paper here). Their MD simulations examine how this process depends on an electric field across the gap, showing that there is a critical field strength at which the inhibitory influence of surface tension is overcome by electrostatic pressure. The focus here is mostly on scanning-probe-microscope-based experiments and technologies such as dip-pen nanolithography, but one can presumably imagine charging mechanisms for drawing water columns inside otherwise hydrophobic cavities in biomolecules. I have no idea whether such things are observed in nature…
There’s another take on nanoconfined water in a paper by Matthew Lane and colleagues at Sandia (J. M. D. Lane et al., Langmuir 24, 5209-5212; 2008 – paper here). They study the dynamics of a very thin film of water (submonolayer to bilayer) between two carboxyl-terminated alkanethiol self-assembled monolayers, using MD simulations. The diffusion coefficient of the water decreases as the layers become thinner, down to two orders of magnitude less than the bulk value, but the water remains liquid-like.
David Chandler, working with Adam Willard, has more on the role of solvent fluctuations in his dewetting model of hydrophobic assembly (discussed in earlier posts) in a paper in J. Phys. Chem. B 112, 6187-6192; 2008 (paper here). Fluctuations play a crucial role in the formation of a ‘vapour tunnel’ between two spherical hydrophobic particles, which draws them together. And in the same volume, Peter Rossky and colleagues expand on their work on the mechanism of protein cold denaturation, which I’ve also mentioned previously (C. F. Lopez et al., J. Phys. Chem. B 112, 5961-5967; 2008 – paper here).
Here’s another nice example of bound water playing a crucial role in enzyme function in a paper by Sason Shaik at the Hebrew University of Jerusalem and colleagues (Y. Wang et al., JACS 10.1021/ja711426y). They have looked at the mechanism by which cytochrome P450 StaP catalyses the formation of staurosporine, an antitumour agent, from chromopyrrolic acid. A critical step in this process is the abstraction of a proton from an N-H group on the substrate by an iron-oxo species in the enzyme. This seems to happen with the concerted assistance of two water molecules in the binding site: one shifts the proton onto a nearby histidine residue, and the other takes a proton from the other side of this residue’s side-chain and puts it on the iron-oxo group. Thus, the water molecules here form a hydrogen-bonded proton-relay network.
Rohit Pappu and colleagues at the University of St Louis in Missouri have an interesting study on the conformation of intrinsically disordered proteins, a class of protein that lack well defined 3D structures (H. T. Tran et al., JACS 10.1021/ja710446s – paper here). The common notion seems to be that because these proteins have sequences of low hydrophobicity, they are not tightly bound into compact structures by hydrophobic interactions. But IDPs are not totally random – they do have an ensemble of preferred conformational states. What creates them? The authors use simulations to conclude that these states are not dominated by specific intramolecular interactions in the polypeptide backbone, but from solvent-solute interactions, since water seems to be a generically poor solvent even for these low-hydrophobicity backbones.
More on hydration of poorly folded proteins comes from Supid Chakraborty and Sanjoy Bandyopadhyay at the Indian Institute of Technology in Kharagpur. They have used simulations to look at how the unfolding of the HP-36 subdomain of villin headpiece protein affects the dynamics of the hydration shell (J. Phys. Chem. B 112, 6500-6507; 2008 – paper here). Unfolding turns out to have a strong but quite complex effect on the rotational and translational motions of water in the hydration shell. It seems possible that there are knock-on effects: as one part of the protein unfolds, this can alter the structure and dynamics of hydration water around other segments in a cooperative manner. There’s clearly much more to be done on this interesting but under-investigated issue.
There’s more, as ever, to come when time permits…
Friday, May 9, 2008
Ions and water at interfaces: will they be understood by August?
There has been a lot of debate, some described in earlier posts, about the behaviour of hydroxide and hydronium ions at air-water and hydrophobic interfaces. Some claim the air-water interface is acidic, others that it is basic. So do protons or hydroxide ions get preferentially adsorbed at these interfaces? I’ve been sent a preprint (now published in J. Phys. Chem. C, doi:10.1021/jp800888b; paper here) by Robert Vácha, Ronen Zangi, Jan Engberts and Pavel Jungwirth that casts new light on the issue with simulations of hydroxide hydration near hydrophobic walls in aqueous KOH. They find that rigid walls create strong layering effects and a peak of hydroxide concentration about 5Å from the wall. But when the wall atoms are allowed to vibrate thermally, and when the dispersion interactions are weaker (more like the air-water interface), this structuring tends to get washed out, in some cases completely. Thus it seems one can’t generalize about hydroxide adsorption (or not) at a hydrophobic interface.
Another blow to ‘water structure effects’ at interfaces comes from a paper by Mischa Bonn and colleagues, in Huib Bakker’s group at FOM Amsterdam (M. Sovago et al., Phys. Rev. Lett. 100, 173901; 2008 – paper here). They have investigated the double-peaked vibrational sum-frequency generation (VSFG) spectrum of the O-H bonds of interfacial water in the hydrogen-bonded region, at a lipid-water interface. The two peaks have previously been interpreted as two distinct classes of hydrogen bond, ‘weak’ (ice-like) and ‘strong’ (water-like). But the FOM team, along with others from the Universities of Amsterdam and Utrecht, have evidence from isotope substitution experiments that the double peak is due to vibrational coupling between stretching and bending modes. They therefore conclude that the interfacial water is more homogeneous than has been thought.
How ions affect hydration and hydrophobic interactions is one of the thorniest problems in this field, and I won’t repeat myself by trying to summarize what has been said previously on the issue. Suffice to say that this question is intimately bound up with the notoriously puzzling ion-specific Hofmeister effects on solubility and aggregation of proteins. Neither am I going to make a poor attempt at summarizing the conclusions of a new investigation into these phenomena by Roland Netz and colleagues in Germany and Sweden (D. Horinek et al., Langmuir 24, 1271-1283; 2008 – paper here). Let’s just say that they have used single-molecule AFM experiments and MD simulations to delve into the ion-specific factors, free from complications of bubble nucleation and cavitation that may intervene for extended surfaces. The punchline is that “the most important factor determining ion-specific adsorption at hydrophobic surfaces can best be described as surface-modified ion hydration” – but you’d best read the paper to unpack that. A useful addition to a complicated story.
Lawrence Pratt and coworkers have a preprint exploring the role of dispersion forces on the potential of mean force between methane molecules in water. The find that these attractive methane-water interactions contribute a repulsive term to the pair potential (potential of mean force, pmf) between methanes. They also say that packing effects in the hydration shells make a dominant contribution to this pmf, but not in a way that can be interpreted with a perturbative approach – that is (if I’ve understood this properly), by treating the pmf as a perturbation expansion around this basic term.
Haiping Fang of the Shanghai Institute of Applied Physics and his coworkers have a very nice ‘topical review’ entitled ‘Dynamics of single-file water chains inside nanoscale channels: physics, biological significance and applications’ in J. Phys. D: Appl. Phys. 41, 103002 (2008). Some of the same issues, and some others, are addressed in a recent review by Gerhard Hummer and colleagues: J. C. Rasaiah et al., ‘Water in nonpolar confinement: from proteins to nanotubes and beyond’, Ann. Rev. Phys. Chem. 59, 713-740 (2008).
Finally, a free advert for a RSC Faraday Division discussion meeting on 27-29 August at Heriot-Watt University in Edinburgh entitled ‘Water: From Interfaces to the Bulk’ (see details here.). The announcement says that this meeting “plans to achieve a unification of views towards the goal of understanding the microscopic structure and behaviour of condensed phases of water at interfaces and progressing into the bulk.” I wish I could be there.
Another blow to ‘water structure effects’ at interfaces comes from a paper by Mischa Bonn and colleagues, in Huib Bakker’s group at FOM Amsterdam (M. Sovago et al., Phys. Rev. Lett. 100, 173901; 2008 – paper here). They have investigated the double-peaked vibrational sum-frequency generation (VSFG) spectrum of the O-H bonds of interfacial water in the hydrogen-bonded region, at a lipid-water interface. The two peaks have previously been interpreted as two distinct classes of hydrogen bond, ‘weak’ (ice-like) and ‘strong’ (water-like). But the FOM team, along with others from the Universities of Amsterdam and Utrecht, have evidence from isotope substitution experiments that the double peak is due to vibrational coupling between stretching and bending modes. They therefore conclude that the interfacial water is more homogeneous than has been thought.
How ions affect hydration and hydrophobic interactions is one of the thorniest problems in this field, and I won’t repeat myself by trying to summarize what has been said previously on the issue. Suffice to say that this question is intimately bound up with the notoriously puzzling ion-specific Hofmeister effects on solubility and aggregation of proteins. Neither am I going to make a poor attempt at summarizing the conclusions of a new investigation into these phenomena by Roland Netz and colleagues in Germany and Sweden (D. Horinek et al., Langmuir 24, 1271-1283; 2008 – paper here). Let’s just say that they have used single-molecule AFM experiments and MD simulations to delve into the ion-specific factors, free from complications of bubble nucleation and cavitation that may intervene for extended surfaces. The punchline is that “the most important factor determining ion-specific adsorption at hydrophobic surfaces can best be described as surface-modified ion hydration” – but you’d best read the paper to unpack that. A useful addition to a complicated story.
Lawrence Pratt and coworkers have a preprint exploring the role of dispersion forces on the potential of mean force between methane molecules in water. The find that these attractive methane-water interactions contribute a repulsive term to the pair potential (potential of mean force, pmf) between methanes. They also say that packing effects in the hydration shells make a dominant contribution to this pmf, but not in a way that can be interpreted with a perturbative approach – that is (if I’ve understood this properly), by treating the pmf as a perturbation expansion around this basic term.
Haiping Fang of the Shanghai Institute of Applied Physics and his coworkers have a very nice ‘topical review’ entitled ‘Dynamics of single-file water chains inside nanoscale channels: physics, biological significance and applications’ in J. Phys. D: Appl. Phys. 41, 103002 (2008). Some of the same issues, and some others, are addressed in a recent review by Gerhard Hummer and colleagues: J. C. Rasaiah et al., ‘Water in nonpolar confinement: from proteins to nanotubes and beyond’, Ann. Rev. Phys. Chem. 59, 713-740 (2008).
Finally, a free advert for a RSC Faraday Division discussion meeting on 27-29 August at Heriot-Watt University in Edinburgh entitled ‘Water: From Interfaces to the Bulk’ (see details here.). The announcement says that this meeting “plans to achieve a unification of views towards the goal of understanding the microscopic structure and behaviour of condensed phases of water at interfaces and progressing into the bulk.” I wish I could be there.
Friday, May 2, 2008
Life in the cold
[This is a slightly more considered summary of recent work on cryoprotectants, which appears as my Crucible column in the May issue of Chemistry World. The links to the papers discussed can be found in the previous blog entry below.]
When the going gets tough, the tough get sweet. There are many physiological responses to cold conditions, from goose pimples (useless for humans, handier for hairier beasts) to the famous antifreeze proteins of fish. But one of the common strategies for insects is to fill their cells with sugar. It’s still something of a mystery why this helps.
Cold poses diverse threats to life. Ice crystals in the body can simply rupture cell walls, which is why frozen strawberries thaw to a mush. And below about –20 degC protein molecules themselves start to unravel, a process called cold denaturation. That’s not well understood yet either, although a recent paper[1] suggests it involves weakening of the force between hydrophobic (water-repelling) parts of proteins that normally binds the folded form in place.
Sugars such as fructose and trehalose, as well as polyols such as glycerol and ethylene glycol, are manufactured seasonally by insects as cryoprotectants, just as we put antifreeze in our car radiators as winter draws near. Over winter, up to a fifth of the mass of some insects may consist of these substances. One consequence of cell fluid rich in sugar is simple depression of water’s freezing point. But that won’t get you very far into a bitter winter – typically, the depression will be only a few degrees, whereas the Arctic willow gall, flooded with glycerol, will survive temperatures of –66 degC. It’s thought that the cryoprotectants are doing something else here too.
Surviving the cold isn’t always about not getting frozen; sometimes there’s no avoiding it, and cryoprotectants then seem to act as freeze-tolerance rather than freeze-avoidance agents. It’s not only insects that do this: some frogs can survive being frozen solid if they fill their cells with glucose. The sugars and polyols seem to interact with cell water to protect delicate proteins and membranes – but no one is sure how.
Minoru Sakurai of the Tokyo Institute of Technology and his coworkers have shed some light on this through studies of the African midge Polypedilum vanderplanki[2]. They’ve studied not freezing as such, but an environmental stress more common in Africa which has similar consequences: dehydration. Dried larvae of the midge can enter a state called anhydrobiosis, in which they show no metabolic activity but can recover viability when water becomes available. They do this by generating trehalose.
There have been two suggestions for the protective mechanism: either the water is substituted by the sugar, or the sugar promotes the formation of a glassy cell matrix rather than ice crystals. The Japanese team thinks that in fact both are true. They find that the sugar forms hydrogen bonds with the lipids in cell membranes, replacing a shell of hydration water and preventing the membranes from becoming rigid. But the larvae also undergo a distinct glass transition as they are slowly dried. The glass is not pure trehalose, but is peppered with other components, such as proteins, that might help to disrupt crystallization.
How, though, does a shell of sugar or polyol protect a protein when water cannot? It seems that cryoprotectants can stabilize proteins against unfolding, but whether this comes from direct protein-sugar interactions or some kind of sugar-induced modification of water structure isn’t clear. Martina Havenith at the Ruhr University of Bochum and her colleagues recently reported signs that the latter might play a role[3]. Using terahertz spectroscopy, they found that the dynamics of water molecules are disturbed a remarkably long distance away from dissolved sugars – up to about 5-7 Å for trehalose and lactose. These perturbations are stronger and longer-ranged for disaccharides than for the monosaccharide glucose, which would support the notion that cryoprotection (which disacchardides do better) is tied up with the sugar’s ability to slow down the water motions and promote a pseudo-glassy state.
Findings by Giovanni Strambini and coworkers at the Consiglio Nazionale delle Ricerche in Pisa, Italy, could be seen to lend support to this idea. The Italian team have asked how cryoprotectants do their job if ice actually begins to form. They used fluorescence spectroscopy to study the stabilization of a protein called azurin by sugars and polyols (sucrose, trehalose, sorbitol, glycerol) in ice-water mixtures[4]. It seems none of these molecules offers strong protection against ice formation, although trehalose ‘tries hardest’: as ice appears, the protein is increasingly prone to unfold. So the cryoprotectants don’t make the native protein significantly more thermodynamically stable. Instead, the researchers think that they somehow cajole the protein to stay folded in the liquid until the whole system becomes a sluggish glass and unfolding is then simply too slow – a kinetic rather than thermodynamic effect.
So there is an emerging picture, albeit a complex one. The cryoprotectants could have a dual role. First they remodel biomolecular hydration shells, retarding water and maybe suppressing the loss of crucial protein-folding forces. Then they eventually promote the formation of a glassy matrix rather than an icy one, arresting the biomolecular structures in recoverable suspended animation. That’s clever work for a spoonful of sugar.
References
1. C. L. Dias et al., Phys. Rev. Lett. 100, 118101 (2008).
2. M. Sakurai et al., Proc. Natl Acad. Sci. USA 105, 5093-5098 (2008).
3. M. Heyden et al., J. Am. Chem. Soc. doi:10.1021/ja0781083.
4. G. B. Strambini et al., J. Phys. Chem. B 112, 4372-4380 (2008).
When the going gets tough, the tough get sweet. There are many physiological responses to cold conditions, from goose pimples (useless for humans, handier for hairier beasts) to the famous antifreeze proteins of fish. But one of the common strategies for insects is to fill their cells with sugar. It’s still something of a mystery why this helps.
Cold poses diverse threats to life. Ice crystals in the body can simply rupture cell walls, which is why frozen strawberries thaw to a mush. And below about –20 degC protein molecules themselves start to unravel, a process called cold denaturation. That’s not well understood yet either, although a recent paper[1] suggests it involves weakening of the force between hydrophobic (water-repelling) parts of proteins that normally binds the folded form in place.
Sugars such as fructose and trehalose, as well as polyols such as glycerol and ethylene glycol, are manufactured seasonally by insects as cryoprotectants, just as we put antifreeze in our car radiators as winter draws near. Over winter, up to a fifth of the mass of some insects may consist of these substances. One consequence of cell fluid rich in sugar is simple depression of water’s freezing point. But that won’t get you very far into a bitter winter – typically, the depression will be only a few degrees, whereas the Arctic willow gall, flooded with glycerol, will survive temperatures of –66 degC. It’s thought that the cryoprotectants are doing something else here too.
Surviving the cold isn’t always about not getting frozen; sometimes there’s no avoiding it, and cryoprotectants then seem to act as freeze-tolerance rather than freeze-avoidance agents. It’s not only insects that do this: some frogs can survive being frozen solid if they fill their cells with glucose. The sugars and polyols seem to interact with cell water to protect delicate proteins and membranes – but no one is sure how.
Minoru Sakurai of the Tokyo Institute of Technology and his coworkers have shed some light on this through studies of the African midge Polypedilum vanderplanki[2]. They’ve studied not freezing as such, but an environmental stress more common in Africa which has similar consequences: dehydration. Dried larvae of the midge can enter a state called anhydrobiosis, in which they show no metabolic activity but can recover viability when water becomes available. They do this by generating trehalose.
There have been two suggestions for the protective mechanism: either the water is substituted by the sugar, or the sugar promotes the formation of a glassy cell matrix rather than ice crystals. The Japanese team thinks that in fact both are true. They find that the sugar forms hydrogen bonds with the lipids in cell membranes, replacing a shell of hydration water and preventing the membranes from becoming rigid. But the larvae also undergo a distinct glass transition as they are slowly dried. The glass is not pure trehalose, but is peppered with other components, such as proteins, that might help to disrupt crystallization.
How, though, does a shell of sugar or polyol protect a protein when water cannot? It seems that cryoprotectants can stabilize proteins against unfolding, but whether this comes from direct protein-sugar interactions or some kind of sugar-induced modification of water structure isn’t clear. Martina Havenith at the Ruhr University of Bochum and her colleagues recently reported signs that the latter might play a role[3]. Using terahertz spectroscopy, they found that the dynamics of water molecules are disturbed a remarkably long distance away from dissolved sugars – up to about 5-7 Å for trehalose and lactose. These perturbations are stronger and longer-ranged for disaccharides than for the monosaccharide glucose, which would support the notion that cryoprotection (which disacchardides do better) is tied up with the sugar’s ability to slow down the water motions and promote a pseudo-glassy state.
Findings by Giovanni Strambini and coworkers at the Consiglio Nazionale delle Ricerche in Pisa, Italy, could be seen to lend support to this idea. The Italian team have asked how cryoprotectants do their job if ice actually begins to form. They used fluorescence spectroscopy to study the stabilization of a protein called azurin by sugars and polyols (sucrose, trehalose, sorbitol, glycerol) in ice-water mixtures[4]. It seems none of these molecules offers strong protection against ice formation, although trehalose ‘tries hardest’: as ice appears, the protein is increasingly prone to unfold. So the cryoprotectants don’t make the native protein significantly more thermodynamically stable. Instead, the researchers think that they somehow cajole the protein to stay folded in the liquid until the whole system becomes a sluggish glass and unfolding is then simply too slow – a kinetic rather than thermodynamic effect.
So there is an emerging picture, albeit a complex one. The cryoprotectants could have a dual role. First they remodel biomolecular hydration shells, retarding water and maybe suppressing the loss of crucial protein-folding forces. Then they eventually promote the formation of a glassy matrix rather than an icy one, arresting the biomolecular structures in recoverable suspended animation. That’s clever work for a spoonful of sugar.
References
1. C. L. Dias et al., Phys. Rev. Lett. 100, 118101 (2008).
2. M. Sakurai et al., Proc. Natl Acad. Sci. USA 105, 5093-5098 (2008).
3. M. Heyden et al., J. Am. Chem. Soc. doi:10.1021/ja0781083.
4. G. B. Strambini et al., J. Phys. Chem. B 112, 4372-4380 (2008).
Tuesday, April 8, 2008
Surviving the winter
How do some organisms survive dehydration? In the state called anhydrobiosis, such organisms exhibit a kind of suspended animation, showing no apparent metabolic activity but reviving when rehydrated. Two explanations for this behaviour have been proposed: replacement of water by another medium, such as sugars, and the formation of a glassy matrix (vitrification). Takashi Okuda of the National Institute of Agrobiological Sciences in Tsukuba, Japan, and his colleagues have reported evidence that, at least for the case of the African midge Polypedilum vanderplanki, both these hypotheses seem to apply [M. Sakurai et al., PNAS 105, 5093-5098; 2008 – paper here]. They find that anhydrobiotic larvae of this insect are in a glassy state in which much of the water is replaced with trehalose. They think that the glassy matrix is a mixture of trehalose with other components, such as highly hydrophilic proteins. The big question is surely how the organisms get into and out of the glassy state: if moisture uptake or high temperature turns the sugar glass rubbery, the larvae lose their viability.
The production of trehalose and other compatible solutes is of course one of the common mechanisms of freeze-tolerance. Cold conditions threaten organisms not just because of the physical disruption caused by ice crystal formation, but because proteins may denature below about minus 20 Centigrade. The mechanism of cold denaturation has been much debated. Cristiano Dias of the University of Montreal and colleagues propose [C. L. Dias et al., Phys. Rev. Lett. 100, 118101; 2008 – paper here] that it shares characteristics with pressure-induced denaturation, which Gerhard Hummer and his coworkers have previously attributed to the destabilization of hydrophobic contacts in favour of solvent-separated ones [G. Hummer et al., PNAS 95, 1552; 1998]. In other words, the idea is that both effects are all about the temperature- and pressure-dependence of the hydrophobic interaction. Dias and colleagues support this conclusion with 2D molecular-dynamics simulations, which suggest that at low temperatures water molecules in a protein’s hydration shell hydrogen-bond more strongly than those in the bulk.
It’s a bit of a month for studies of freeze/dehydration tolerance. Martina Havenith at Bochum and colleagues have used terahertz spectroscopy to show surprisingly long-ranged correlations between sub-picosecond dynamics in water close to and further from dissolved cabohydrates (trehalose, lactose, glucose) [M. Heyden et al., JACS doi:10.1021/ja0781083– paper here]. That is, the effects persist over about 5-7 Å, suggesting that this is in effect the width of the hydration shell for these sugars. The perturbations are stronger for the disaccharides than the monosaccharide (here the influence extends only to about 3-4 Å), which the authors connect with the stronger cryoprotection activity of the former – that is, it would support the notion that this protection is conferred by a disruption (retardation) of the solvent dynamics.
And Giovanni Strambini and coworkers at the Consiglio Nazionale delle Ricerche in Pisa, Italy, have used fluorescence spectroscopy to study the stabilization of the azurin protein by sugars and polyols in ice [G. B. Strambini et al., J. Phys. Chem. B 112, 4372-4380; 2008 – paper here]. One question they have set out to address is: to what extent are the cryoprotectants defending against low temperature per se, and to what extent against ice formation? In fact, none of the molecules (sucrose, trehalose, sorbitol, glycerol) seems to fully protect against ice formation, although trehalose ‘tries hardest’. It seems that the role of the cryoprotectants is not to increase the thermodynamic stability of the native fold per se, but to help hold it together specifically in the liquid phase. Curiously, some of them do this less effectively at minus 15 C than at lower temperatures – which seems to be because once it gets cold enough, denaturation becomes kinetically rather than thermodynamically controlled: unfolding is simply too slow. There is an interesting story building here.
More on low-temperature protein dynamics comes from Jeremy Smith and his coworkers Vandana Kurkal-Siebert and Ritesh Agarwal at Heidelberg [Phys. Rev. Lett. 100, 138102; 2008 – paper here]. They have used MD simulations to look at interprotein dynamical interactions in hydrated crystals of carboxymyoglobin, a kind of proxy for protein-protein interactions more generally. In fully hydrated crystals there is a dynamical transition at around 240 K due to intermolecular fluctuations, whereas no such behaviour is seen for low hydration. This is reminiscent of the 220 K intramolecular dynamical transition seen in proteins, which resembles (but may not in fact be – see below) a glass transition. If I’ve understood this properly, the idea is that the (diffusive) intermolecular motions in the proteins must therefore be mediated (activated) by those in the hydration shells.
The dynamics of proteins in this pseudo-glassy state below about 220 K do indeed seem to get very closely coupled – perhaps ‘slaved’ – to the similar non-Arrhenius dynamics of the solvent. Now Martin Weik and a host of others have confirmed this coupling using neutron scattering [K. Wood et al., JACS 10.1021/ja710526r – paper here]. They find that below the transition temperature, the protein (here maltose binding protein) is structurally arrested in a glassy solvent cage, while at around 220 K ‘fast’ protein motions are reawakened at precisely the same time as hydration water starts to show diffusive translational motion. In a nutshell, as the authors put it, “the protein dynamical transition is correlated with relaxation of the protein H-bond network, which, in turn, is associated with the onset of water translational diffusion.”
Sow-Hsin Chen at MIT and his coworkers have previously related this dynamical transition to that in pure water predicted on the basis of a liquid-liquid transition at high pressure, which creates a kind of ‘ghost’ of the transition called the Widom line at lower pressures (see S.-H. Chen et al., PNAS 103, 9012; 2006). They now report further evidence, from SANS studies of water in mesoporous silica (MCM-41), of a change in the structure and dynamics of water at around 235 K due to crossing of the Widom line [D. Liu et al., J. Phys. Chem. B 112, 4309-4312; 2008 – paper here] .
Finally (for now), Roland Netz and colleagues at the Technical University of Munich have attempted to simplify studies of the hydrophobic attraction, which has typically involved mesoscopic surfaces on which many length scales can be important, by investigating the force required to peel a single, mildly hydrophobic peptide from a diamond surface [D. Horinek et al., PNAS 105, 2842-2847; 2008 – paper here]. They compare their experimental results using atomic force spectroscopy with simulation, to try to quantify the relative contributions of dispersion forces between the two surfaces and ‘water structure’ effects. They find that all the individual pairwise interactions between the peptide, the surface and the solvent are larger than the total desorption energy, but opposite in sign while being of similar magnitude, so that they almost cancel out.
The production of trehalose and other compatible solutes is of course one of the common mechanisms of freeze-tolerance. Cold conditions threaten organisms not just because of the physical disruption caused by ice crystal formation, but because proteins may denature below about minus 20 Centigrade. The mechanism of cold denaturation has been much debated. Cristiano Dias of the University of Montreal and colleagues propose [C. L. Dias et al., Phys. Rev. Lett. 100, 118101; 2008 – paper here] that it shares characteristics with pressure-induced denaturation, which Gerhard Hummer and his coworkers have previously attributed to the destabilization of hydrophobic contacts in favour of solvent-separated ones [G. Hummer et al., PNAS 95, 1552; 1998]. In other words, the idea is that both effects are all about the temperature- and pressure-dependence of the hydrophobic interaction. Dias and colleagues support this conclusion with 2D molecular-dynamics simulations, which suggest that at low temperatures water molecules in a protein’s hydration shell hydrogen-bond more strongly than those in the bulk.
It’s a bit of a month for studies of freeze/dehydration tolerance. Martina Havenith at Bochum and colleagues have used terahertz spectroscopy to show surprisingly long-ranged correlations between sub-picosecond dynamics in water close to and further from dissolved cabohydrates (trehalose, lactose, glucose) [M. Heyden et al., JACS doi:10.1021/ja0781083– paper here]. That is, the effects persist over about 5-7 Å, suggesting that this is in effect the width of the hydration shell for these sugars. The perturbations are stronger for the disaccharides than the monosaccharide (here the influence extends only to about 3-4 Å), which the authors connect with the stronger cryoprotection activity of the former – that is, it would support the notion that this protection is conferred by a disruption (retardation) of the solvent dynamics.
And Giovanni Strambini and coworkers at the Consiglio Nazionale delle Ricerche in Pisa, Italy, have used fluorescence spectroscopy to study the stabilization of the azurin protein by sugars and polyols in ice [G. B. Strambini et al., J. Phys. Chem. B 112, 4372-4380; 2008 – paper here]. One question they have set out to address is: to what extent are the cryoprotectants defending against low temperature per se, and to what extent against ice formation? In fact, none of the molecules (sucrose, trehalose, sorbitol, glycerol) seems to fully protect against ice formation, although trehalose ‘tries hardest’. It seems that the role of the cryoprotectants is not to increase the thermodynamic stability of the native fold per se, but to help hold it together specifically in the liquid phase. Curiously, some of them do this less effectively at minus 15 C than at lower temperatures – which seems to be because once it gets cold enough, denaturation becomes kinetically rather than thermodynamically controlled: unfolding is simply too slow. There is an interesting story building here.
More on low-temperature protein dynamics comes from Jeremy Smith and his coworkers Vandana Kurkal-Siebert and Ritesh Agarwal at Heidelberg [Phys. Rev. Lett. 100, 138102; 2008 – paper here]. They have used MD simulations to look at interprotein dynamical interactions in hydrated crystals of carboxymyoglobin, a kind of proxy for protein-protein interactions more generally. In fully hydrated crystals there is a dynamical transition at around 240 K due to intermolecular fluctuations, whereas no such behaviour is seen for low hydration. This is reminiscent of the 220 K intramolecular dynamical transition seen in proteins, which resembles (but may not in fact be – see below) a glass transition. If I’ve understood this properly, the idea is that the (diffusive) intermolecular motions in the proteins must therefore be mediated (activated) by those in the hydration shells.
The dynamics of proteins in this pseudo-glassy state below about 220 K do indeed seem to get very closely coupled – perhaps ‘slaved’ – to the similar non-Arrhenius dynamics of the solvent. Now Martin Weik and a host of others have confirmed this coupling using neutron scattering [K. Wood et al., JACS 10.1021/ja710526r – paper here]. They find that below the transition temperature, the protein (here maltose binding protein) is structurally arrested in a glassy solvent cage, while at around 220 K ‘fast’ protein motions are reawakened at precisely the same time as hydration water starts to show diffusive translational motion. In a nutshell, as the authors put it, “the protein dynamical transition is correlated with relaxation of the protein H-bond network, which, in turn, is associated with the onset of water translational diffusion.”
Sow-Hsin Chen at MIT and his coworkers have previously related this dynamical transition to that in pure water predicted on the basis of a liquid-liquid transition at high pressure, which creates a kind of ‘ghost’ of the transition called the Widom line at lower pressures (see S.-H. Chen et al., PNAS 103, 9012; 2006). They now report further evidence, from SANS studies of water in mesoporous silica (MCM-41), of a change in the structure and dynamics of water at around 235 K due to crossing of the Widom line [D. Liu et al., J. Phys. Chem. B 112, 4309-4312; 2008 – paper here] .
Finally (for now), Roland Netz and colleagues at the Technical University of Munich have attempted to simplify studies of the hydrophobic attraction, which has typically involved mesoscopic surfaces on which many length scales can be important, by investigating the force required to peel a single, mildly hydrophobic peptide from a diamond surface [D. Horinek et al., PNAS 105, 2842-2847; 2008 – paper here]. They compare their experimental results using atomic force spectroscopy with simulation, to try to quantify the relative contributions of dispersion forces between the two surfaces and ‘water structure’ effects. They find that all the individual pairwise interactions between the peptide, the surface and the solvent are larger than the total desorption energy, but opposite in sign while being of similar magnitude, so that they almost cancel out.
Wednesday, March 26, 2008
Chemistry vs geometry
Pablo Debenedetti and colleagues have carried out precisely the kind of study that is needed to tease apart the various factors that might be at play in hydrophobic association of proteins (N. Giovambattista et al., PNAS 105, 2274-2279; 2008 – paper here). One can anticipate that the potential for effects such as abrupt drying transitions as the two surfaces approach is affected both by surface chemistry – by the distribution of hydrophilic and hydrophobic groups – and by geometry. Certainly, both have been implicated as playing a role in how real proteins behave, as for example in the Berne group’s study of protein associations for BphC and melittin (Liu et al., Nature 437, 159-162; 2005; Zhou et al., Science 305, 1605-1609; 2004). Melittin monomers enclose a tubelike space, for example, whereas BphC is slablike. Moreover, melittin, like many proteins, has a rough surface with concavities. To decouple the effects, Pablo and colleagues have simulated the association of a mutated melittin dimer in which the distribution of hydrophobic and hydrophilic groups is retained but the surface is artificially flattened. The results suggest that the flattened melittin behaves as an intermediate case between ideal, flat hydrophobic and hydrophilic surfaces, and that the drying seen in the case of ‘real’ melittin happens only at very small separations (about one intervening water layer) for the flattened case, being localized to a central region where an apolar residue resides. It can be suppressed by replacing that residue. In other words, drying seen for ideal hydrophobic plates is probably stronger than it is for real proteins, where it is likely to be highly sensitive to small variations in surface chemistry.
Michael Geisler and colleagues at the Technical University of Munich have looked at Hofmeister effects in the adhesion of spider silk proteins to a solid surface, using single-molecule AFM force spectroscopy (Langmuir 24, 1350-1355; 2008 – paper here). They find that the desorption forces follow the Hofmeister series, but can’t yet develop a clear interpretation of what is going on. The hydrophobicity of the silk protein also plays a part: ions that stabilize adhesion do so less when the protein is less hydrophobic, ‘indicating that hydrophobic and Hofmeister effects are closely related’ – but how?
Dusan Bratko and Alenka Luzar have attempted to unravel the much vexed question of how dissolved gases affect the hydrophobic interaction (Langmuir 24, 1247-1253; 2008 – paper here). They have used simulations to look at how various gases influence water structure close to a hydrophobic surface, and solvation forces between two such surfaces. They say that although there does seem to be accumulation of dissolved gas at the interface, it doesn’t have a big effect either on putative water depletion or on solvation forces – something that several experiments seem to bear out. One of the nice aspects of this work is that it enables the authors to make a link between capillary evaporation of pure water induced by hydrophobic confinement and evaporation nucleated by an excess of dissolved gas at the interface – two things that are sometimes not so clearly distinguished. But the simulations can’t follow the possible formation of nanobubbles and the effect this might have on the hydrophobic interaction.
Finally, I have good reason to think that my recent Essay in Nature on water (here) might be seen by some as an endorsement of the ‘new view’ of water structure championed by Anders Nilsson and Lars Pettersson. It’s not, as I think is clear if you read carefully. I merely point out that, first, it is remarkable that such fundamental disagreements about water are still occurring (I know, of course, that Anders and Lars’ idea has been strongly criticized), and secondly, that the implications are rather more far-reaching than might be naively supposed. I must apologize, incidentally, for giving the impression that the experimental work on which their new model is based was done by Lars at Stockholm, rather than by Anders at Stanford.
Michael Geisler and colleagues at the Technical University of Munich have looked at Hofmeister effects in the adhesion of spider silk proteins to a solid surface, using single-molecule AFM force spectroscopy (Langmuir 24, 1350-1355; 2008 – paper here). They find that the desorption forces follow the Hofmeister series, but can’t yet develop a clear interpretation of what is going on. The hydrophobicity of the silk protein also plays a part: ions that stabilize adhesion do so less when the protein is less hydrophobic, ‘indicating that hydrophobic and Hofmeister effects are closely related’ – but how?
Dusan Bratko and Alenka Luzar have attempted to unravel the much vexed question of how dissolved gases affect the hydrophobic interaction (Langmuir 24, 1247-1253; 2008 – paper here). They have used simulations to look at how various gases influence water structure close to a hydrophobic surface, and solvation forces between two such surfaces. They say that although there does seem to be accumulation of dissolved gas at the interface, it doesn’t have a big effect either on putative water depletion or on solvation forces – something that several experiments seem to bear out. One of the nice aspects of this work is that it enables the authors to make a link between capillary evaporation of pure water induced by hydrophobic confinement and evaporation nucleated by an excess of dissolved gas at the interface – two things that are sometimes not so clearly distinguished. But the simulations can’t follow the possible formation of nanobubbles and the effect this might have on the hydrophobic interaction.
Finally, I have good reason to think that my recent Essay in Nature on water (here) might be seen by some as an endorsement of the ‘new view’ of water structure championed by Anders Nilsson and Lars Pettersson. It’s not, as I think is clear if you read carefully. I merely point out that, first, it is remarkable that such fundamental disagreements about water are still occurring (I know, of course, that Anders and Lars’ idea has been strongly criticized), and secondly, that the implications are rather more far-reaching than might be naively supposed. I must apologize, incidentally, for giving the impression that the experimental work on which their new model is based was done by Lars at Stockholm, rather than by Anders at Stanford.
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