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.