Meanwhile, Pannuru Venkatesu at the University of Delhi and colleagues have studied the effect of denaturants (urea, GdnHCl) and osmolytes (TMAO, betaine, sucrose and others) on the activity of an enzyme, specifically alpha-chymotrypsin (P. Attri et al., J. Phys. Chem. B 114, 1471; 2010 – paper here). They measure the stability of the enzyme as reflected in the Gibbs free energy of unfolding changes, and the enthalpy change, on addition of cosolvent. These variables increase with increasing osmolyte concentration, and decrease on addition of denaturant – which is, I guess, what one would anticipate. CD spectroscopy suggests that these effects are manifested via changes in beta-helix stability. The osmolytes do not, however, seem to affect enzyme activity. In contrast, and consistent with the study above, denaturants seem to act by binding to the enzyme surface, and induce sufficient structural disruption to reduce activity virtually to zero.
And Yi Qin Gao and colleagues at Texas A&M have used MD simulations to look at the effects of urea, tetramethyl urea (TMU) and the osmolyte trimethylamine N-oxide (TMAO) on the structure of water and dissolved proteins (H. Wei et al., J. Phys. Chem. B 114, 557; 2010 – paper here). TMAO weakens interactions between amide carbonyls on model peptides and water, while urea and TMU strengthen them. Consistent with previous studies, they find a direct interaction between urea and the peptides (via the carbonyls). But they also find evidence of a role for indirect effects on denaturation, whereby the cosolvents alter the hydrophobic interaction via changes to the structure and dynamics of water. They find that a peptide fragment of a G protein unfolds by step-by-step breaking of its native hydrogen bonds, coupled to the formation of water-carbonyl bonds.
Similar territory is explored by Feng Guo and Joel Friedman at the Albert Einstein College of Medicine, who have used vibronic sideband luminescence spectroscopy of a gadolinium(III) probe ion to explore changes in hydrogen bonding between hydration waters of a protein induced by osmolytes (J. Phys. Chem. B 113, 16632; 2009 – paper here). They say that while urea initially weakens hydrogen bonding in the hydration layer, polyol osmolytes such as trehalose, sucrose and glucose enhance it. But they argue that as the concentration of urea increases, it actually enhances water occupancy within the protein and hydrogen bonding in the hydration layer, and that this is the first step in the urea-induced unfolding process. There is a delicate balance here, they say, between entropic effects that favour water penetration of the protein and enthalpic effects that favour a robust hydrogen-bonded hydration network. If I understand the argument correctly, the latter dominates for osmolytes and accounts for the stabilization of the compact folded structure in that case.
The nature of the hydrated hydrogen ion has been much debated. Christopher Reed and colleagues at UC Riverside have investigated the issue using IR spectroscopy, and argue that the best description is neither an Eigen ion (H9O4+) nor a Zundel ion (H5O2+), but the species H13O6+, containing a delocalized proton in the central O-H-O group (E. S. Stoyanov et al., JACS 10.1021/ja9101826 – paper here).
Greg Voth and Takefumi Yamashita have meanwhile investigated the nature of hydrated protons near lipid membranes, using MS-EVB calculations (J. Phys. Chem. B 114, 592; 2010 – paper here). They are interested in clarifying the proposed proton-antenna effect whereby lipid membranes collect protons and shuttle them by lateral diffusion to membrane proteins such as ATP synthase. They confirm this picture, saying that the effect arises because of the stabilization of the hydrated proton by the lipid phosphate groups: a Zundel-like cation bridges phosphate and carbonyl groups. Diffusion of protons within the interface region is significantly slower than it is in the bulk.
Voth, along with Noam Agmon and Hanning Chen, also has a paper on the kinetics of proton transport in pure water (J. Phys. Chem. B 114, 333; 2010 – paper here). The calculations support the idea that the migration of the proton (in effect, of the centre of excess charge) depends on significant reorganization of (perhaps up to 20!) surrounding water molecules.
Mischa Bonn and colleagues at FOM in the Netherlands have used a microfluidic device to study changes in proton mobility near hydrophobic surface (JACS ja9083094 – paper here). They figured that if water molecule reorientation is, as posited, crucial to rapid proton migration, then the slower reorientation of waters near hydrophobic groups seen previously by Rezus and Bakker (Phys. Rev. Lett. 99, 148301; 2007) should have a significant effect on proton transport in such an environment. It’s a very neat experiment: laminar flow in the device means that fluorescein fluorescence in one half of the microfluidic channel may be quenched by lateral proton transport from the other half down a pH gradient. The proton diffusion decreases by an order of magnitude when the hydrophobe tetramethylurea is added, which is consistent with (if not perhaps definitive support for) the hypothesis.
The diffusion of water molecules at lipid surfaces, meanwhile, has been investigated experimentally by Ravinath Kausik and Songi Han at UCSB, using Overhauser dynamic nuclear polarization of the proton NMR signal (JACS 131, 18254; 2009 – paper here). At this stage this is largely a demonstration of the feasibility of the technique, which they hope will also be applicable to the study of solvent dynamics in the hydration shells of macromolecules.
Further along this paper trail, it is the mobility of water molecules in the hydration shells of peptides that is the subject of a simulation study by Charusita Chakravarty at the Indian Institute of Technology in Delhi and colleagues (M. Agarwal et al., J. Phys. Chem. B 114, 651; 2010 – paper here). They consider two small peptides: a 16-residue beta-hairpin structure, and deca-alanine. They see layering structure in the water extending at least 10 Å from the peptide surfaces, and the energetics and dynamics are significantly perturbed relative to the bulk: the discussion is couched mostly in terms of the ‘tagged potential energy’, said to be equivalent to the binding energy of an individual water molecule at a particular location at a given instant in time. This is typically 10-15 percent lower in the innermost hydration layer than in the bulk. But I’m not entirely clear what that implies: does it make diffusion faster or slower? (Surely the latter, but that’s not obvious from this measure.)
In the previous post I mentioned work by Ronen Zangi challenging the notion of ions as structure-makers and structure-breakers. Martina Havenith at Bochum and her colleagues have now raised further problems for this issue, using THz spectroscopy of salt solutions (D. A. Schmidt et al., JACS 131, 18512 (2009) – paper here). They say that the results suggest all ions can be considered simply as defects in the H-bonded network, and so can’t be regarded as either chaotropes or kosmotropes. It turns out that the data can be understood using an appealingly simple model in which the ions undergo damped harmonic oscillations – ‘rattling’ – within the water network. In other words, at least the fast (sub-picosecond) ion motions are essentially decoupled from the dynamics of the network.
One suggested mechanisms of pressure-induced denaturation is the penetration of water into the hydrophobic core. This notion is investigated in simulations by Takashi Imai and Yuji Sugita at RIKEN in Japan (J. Phys. Chem. B jp909701j – paper here). Using ubiquitin as a model case, they look in particular at competing scenarios: does water first penetrate and force the protein to swell, or are the cavities pre-formed by structural fluctuations and then fill with water? They find support for the latter picture: the influx of water stabilizes a pre-existing metastable structure, and drives a distinct transition to a relatively unfolded state.
Harold Sheraga at Cornell and colleagues have probed the nature of hydrophobic interactions as the size of hydrophobic solutes approaches the nanoscale limit (M. Makowski et al., J. Phys. Chem. B jp907794h – paper here). Specifically, they consider the crossover point of around 1 nm at which Lum, Chandler and Weeks (J. Phys. Chem. B 103, 4570; 1999) predicted that the mechanism of the hydrophobic interaction will change from that of small solutes to that of extended surfaces. They calculate the potentials of mean force for large hydrophobes such as adamantine and C60 in water. When two solvation spheres for such large species overlap as they form a dimmer in solution, the water molecules trapped in the concave ‘cleft’ at the edges of the interaction have restricted motion and decreased entropy that offsets any free energy gains from increased solute-particle contact. It seems that fullerenes like this, while too large to be treated as small hydrophobic solutes, are not yet large enough to be considered macroscopic hydrophobic surfaces.
Todd Sformo has sent me a fascinating paper on cold survival strategies of the Alaskan beetle (J. Exp. Biol. 213, 502; 2010 – paper here). He and his coworkers finds that this bug vitrifies at around –76 C, and by that means it can survive cooling of an amazing –150 C. The cryoprotection that supports vitrification is in this case glycerol.
When I was the editor at Nature for Reza Ghadiri’s paper on peptide nanotubes in 1993 (Nature 366; 324), I had to make the decision as something of an act of faith. I remain deeply glad that I did, for the work has stood the test of time. Now Padmanabhan Balaram and colleagues at the Indian Institute of Science have used peptide nanotubes formed from non-cyclic pentamers to study single-file water wires threading through their hydrophobic central channels in molecular crystals (U. S. Raghavender et al., JACS ja9083978 – paper here). This looks like an attractive model system for investigating the relationship between the water structures and the chemical nature of the wall ‘lining’.
Gene Stanley and his coworkers propose a new way of considering the structure of water that involves characterizing the ‘tetrahedral entropy’ associated with the degree of tetrahedral order (P. Kumar et al., PNAS 10.1073/pnas.0911094106 – paper here). They say that this parameter accounts for the specific heat maximum as the Widom line – where there is a cross-over from non-Arrhenius to Arrhenius dynamical behaviour, in general under conditions of supercooling – is crossed.
