Monday, December 8, 2014

What to think about Kauzmann

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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