Friday, November 6, 2009

How do spores survive?

How do bacterial spores survive in a dormant state for years, perhaps in the face of high temperatures or toxic substances? It has been long suspected that the state of water in the cell compartments plays a role. Bertil Halle and his coworkers at Lund have now looked that the state of water in Bacillus subtilis spores using deuterium and oxygen-17 spin relaxation, and they find that the water is not glassy, contrary to some earlier suggestions (E. P. Sunde et al., PNAS 10.1073/pnas.0908712106; paper here). However, the water permeability of the inner membrane is unusually low, providing a barrier to the transport of toxic substances. And some of the key enzymes in the core of the spore seem to be in a relatively dehydrated state, their rotational mobility severely reduced, which might be expected to reduce the tendency of the denatured proteins to aggregate – in other words, the changes in hydration may not provide stabilization against heat-denaturation in itself, but will avoid this becoming an irreversible process.

While most studies of hydration forces between surfaces have tended to focus on hydrophobic surfaces, the nature of the interaction between hydrophilic surfaces is also controversial. It is repulsive, but the reason for this remains debated. There is some suggestion that several mechanisms might act at different length scales – for example, genuine ‘hydration’ effects due to water orientation at moderate separations (between about 0.4 and 0.8 nm), and undulation effects at larger separations. Max Berkowitz at UNC has studied this phenomenon previously using simulations of lipid bilayers (Lu & Berkowitz, J. Chem. Phys. 124, 101101 (2006) and Mol. Phys. 104, 3607 (2006)), but now he and Changsun Eun return to the problem with more accurate simulations in which the lipid head groups are allowed to be mobile (Eun & Berkowitz, J. Phys. Chem. B 113, 13222-13228; 2009 – paper here). They do indeed find three regimes. At short range (<1 nm), the repulsion is dominated by steric van der Waals interactions between the lipid headgroups. They focus mainly on the intermediate-range (1-1.6 nm) interaction, which they argue is due to the free-energy cost of removing waters hydrating the head groups. William Jorgensen has continued his examination of water in protein binding sites, an earlier instance of which was mentioned in the previous post. With Julien Michel and Julian Tirado-Rives, he reports a MD method for determining how water molecules will be situated in binding sites with or without the ligand (J. Phys. Chem. B 113, 13337-13346; 2009 – paper here). The accuracy of the method is shown by comparison with five cases where the crystal structures are known.

Another extension of earlier work: Nicolas Giovambattista, Peter Rossky and Pablo Debenedetti look at how temperature affects the behaviour of water confined between hydrophobic, hydrophilic and heterogeneous nanoscale plates (J. Phys. Chem. B 113, 13723-13734; 2009 - paper here) – the system they considered earlier in Phys. Rev. E 73, 041604 (2006), J. Phys. Chem. C 11, 1323 (2007) and PNAS 105, 2274 (2008). Cooling enables the water to approach the hydrophobic plates more closely, consistent with the expected suppression of the vapour phase. It also blurs the differences in water density between hydrophobic and hydrophilic regions of a heterogeneous surface. This would be consistent with invasion of hydrophobic cavities by water in cold denaturation.

Somewhat related is a study by Ateeque Malani at the Indian Institute of Science in Bangalore and coworkers on the differences in water structure when confined between pairs of two types of hydrophilic surface: hydroxylated silca and mica (J. Phys. Chem. B 113, 13825-13839; 2009 – paper here). While an oscillatory solvation force and a bulk-like H-bond network near the interface are found for silica (these are simulations), the network is disrupted near mica, where there are potassium ions at the surface which are themselves hydrated.

Water diffusion on the surfaces of lipid vesicles has been studied by Ravinath Kausik and Songi Han at UCSB using Overhauser dynamic nuclear polarization of hydrogen-1 NMR (JACS ASAP; paper here). They find diffusion coefficients about half those of bulk water; the key result here is a demonstrating of the feasibility of the technique for obtaining this sort of information. And James Skinner and colleagues at Wisconsin use MD and IR spectroscopy to study water inside reverse micelles (P. A. Pieniazek et al., J. Phys. Chem. B ASAP; paper here). They say that the distance from the surfactant headgroups over which the water becomes bulk-like increases with decreasing micelle size (increasing curvature), eventually becoming larger than the micelle radius. In the smallest micelle (containing 52 water molecules), the water seems to be near-glassy, with very slow rotational relaxation.

Vincent Craig at ANU, now working with Christine Henry, has extended his long-standing studies of the effects of solutes on bubble coalescence. They have looked at the effect on this phenomenon of osmolytes: sucrose and other sugars, and urea (Langmuir 25, 11406-11412; 2009 – paper here). Urea seems to have little effect, but sucrose and other sugars show an inhibiting influence on coalescence. This suggests that, contrary to what one might have been tempted to infer from previous studies on electrolytes, the inhibitory effect does not stem from solute charge. They speculate that concentration gradients close to the bubble-water interface may instead be responsible.

The influence of urea and another osmolyte, trimethylamine-N-oxide on the structure of water and hen egg-white lysozyme are studied using FTIR by Janusz Stangret and colleagues at the Gdansk University of Technology in Poland (A. Panuszko et al., J. Phys. Chem. B ASAP; paper here). Water structure is barely affected by urea, they say, but more strongly perturbed by TMAO, forming stronger and more ‘ordered’ H-bonds. They monitor the protein via the amide I band and suggest that the changes seen there are consistent with changes in water structure, resembling in the case of TMAO changes that are evident on dehydration. This all seems to be presented within the framework of osmolytes exerting indirect effects via their influence on water structure – but I guess one would want to know precisely how the osmolytes interact with the protein itself.

Haiping Fang at Shanghai and colleagues have continued their investigation of water transport through nanochannels. They show how symmetry-breaking of water orientation in a one-dimensional H-bonded chain threading through a carbon nanotube can give rise to spontaneous unidirectional net flux in the absence of any external pressure gradient (R. Wan et al., Phys. Chem. Chem. Phys. 11, 9898-9902; 2009 – paper here doi:10.1039/b907926m). And Haiping also has a paper in PNAS (10.1073/pnas.0902676106; paper here) reporting simulations in which the presence of a single-electron charge in one arm of a Y-shaped carbon nanotube junction can, by flipping the orientation of a water molecule in a single-file chain within the nanotube, reorient the dipoles of the water chains in the other two branches, thus multiplying the single-electron signal. With a suitable arrangement of junctions, it can be multiplied more than twofold.

On a similar topic, Padmanabhan Balaram and colleagues at the Indian Institute of Science use MD simulations to look at the structure of one-dimensional water chains inside the hydrophobic core of a tubular synthetic protein (U. S. Raghavender et al., JACS 131, 15130-15132; 2009 – paper here). They find two distinct states in different peptides: one in which the water molecules are disordered over two possible positions in the chain, the other in which the molecules are perfectly ordered along a sixfold screw axis.

There’s a very neat demonstration of how water in binding sites can be engineered to improve function in a paper on a catalytic antibody by Ian Wilson at Scripps and colleagues (E. W. Debler et al., PNAS 10.1073/pnas.0902700106; paper here). They find that an oriented water molecule in the hydrophobic pocket of the antibody 13G5, which catalyses the cleavage of benzisoxazoles, stabilizies the developing charge on the leaving group. And in a single-residue Glu-to-Ala mutant, a hydrogen-bonded complex involving four water molecules is restructured in a way that enhances still further the rate of the proton transfer involved in the process. A key role for water in another catalytic antibody is reported by Orlando Acevedo at Auburn University in Alabama (J. Phys. Chem. B ASAP; paper here). He looks at the antibody 4B2, which catalyses both a Kemp elimination and an allylic isomerization of an unsatuated ketone. For the former, water molecules in the active site help stabilize the transition state during proton abstraction, while in the latter case the water takes an active role as a proton source. Acevedo suggests that water might be usefully engaged in other designed catalysts to perform this function as a proton donor.

A paper on the behaviour of water and proteins confined in nanoporous (c. 5 nm) silica by Eduardo Reátegui and Alptekin Aksan at Minnesota (J. Phys. Chem. B 113, 13048-13060; 2009 – paper here) contains rather more information than I can easily digest yet. They use FTIR to characterize what is happening to the water, which is of course a slightly blunt tool on its own – certainly, the claim to see liquid-liquid transitions analogous to the putative HDL-LDL transition seems a big one to make on these grounds alone. Changes in the encapsulated proteins, monitored by amide IR bands, seem to mirror those seen in water OH bands, but it’s again not too clear what these actually correspond to in terms of structure or function.

Sotiris Xantheas and Greg Voth, working with Francesc Paesani, have developed an ab initio force field for water that, in MD simulations, provides a good fit for the experimental IR spectra probing H-bond dynamics (J. Phys. Chem. B ASAP; paper here).

The hydrodynamics of water at surfaces has been a controversial topic, and one with some important practical implications. It seems clear that the common no-slip assumption for fluids at solid interfaces doesn’t necessarily hold at the nanoscale. Roland Netz and his coworkers have investigated this for hydrophilic and hydrophobic surfaces using MD simulations (C. Sendner et al., Langmuir 25, 10768-10781; 2009 – paper here). They find something like an inverse square dependence of slip length on contact angle for hydrophobic surfaces, but slip lengths of typically only a few nm for realistic contact angles. This is little affected by dissolved gas at the interface, and the viscosity of the interfacial water is only a few times higher than that of the bulk, with molecular motions being purely diffusive. In contrast, on hydrophilic surface water molecules may become trapped, there is no slip, and the interfacial water viscosity may be enhanced significantly. In the same vein (and consistent with these results), Bharat Bushan and colleagues report measurement of the hydrodynamic forces acting on a glass sphere glued to an AFM tip as it approaches a mica surface (A. Maali et al., Langmuir 25, 12002-12005; 2009 – paper here). They say that the measurements are consistent with a no-slip assumption at both glass and mica surfaces.

Xavier Tadeo and colleagues at the Centro de Investigación Cooperativa bioGUNE in Derio, Spain, have looked at how the anions of the Hofmeister series affect protein stability, using as their test case the IGg binding domain of protein L from Streptoccocal magnus (ProtL) (Biophys. J. 97, 2595-2603; 2009 – paper here). They look at changes in thermostability of a lysine-to-glutamine mutant in the presence sodium salts of sulfate, phosphate, fluoride, nitrate, perchlorate and thiocyanate, and say that the results are consistent with stabilization of the native state by an increase in solution surface tension due to the anions. I’ve not seen the full paper, but I do wonder whether such bulk effects on surface tension can be a reliable guide to what is going on here, without knowing how the ions are partitioned at the protein-solvent interface.

More on Hofmeister effects: Xin Wen and colleagues at CSU at Los Angeles look at the effects of monovalent salts on the activity of the antifreeze protein DAFP-1 from the beetle Dendroides canadensis (S. Wang et al., J. Phys. Chem. B ASAP; paper here). Specifically, they use DSC to monitor how the difference in melting and freezing point of water due to the antifreeze protein is altered by the salts: salting-out seems, as might be expected, to enhance the adsorption of DAFP-1 on the ice surface, thereby boosting its activity.

Henry Ashbaugh at Tulane University has a nice paper on how different sequences of hydrophobic and hydrophilic monomers in a heteropolymer will affect its conformation in aqueous solution (J. Phys. Chem. B 113, 14043-14046; 2009 – paper here). Intermediate segregation of monomer types favours a collapsed conformation, while more strictly alternating monomers favours a random coil.

James Beattie and his coworkers in France and Australia have another paper making the case that the interface of water with air or oil is basic rather than acidic, due to specific adsorption of hydroxide (P. Creux et al., J. Phys. Chem. B ASAP; paper here). This claim is made on the basis of measurements of the zeta potential. I suspect the debate will continue.

There may not be another post from me here for a couple of months, owing to an imminent new arrival in the family. No doubt this means I’ll miss some interesting papers in the interim. But do feel free to send me or tell me of interesting ones (p.ball@btinternet.com). Hope to be back up and running after Christmas – have a good one.

4 comments:

Wavefunction said...

Hi Phil, you seem to have referenced the Balaram water wire paper twice, including in the previous post...you need to post more often!

You may also be interested in a recent editorial by Balaram in the Indian journal Current Science in which he talks about your great book on water:

http://www.ias.ac.in/currsci/oct102009/977.pdf

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