How proteins loosen up

What happens during protein denaturation? One emerging view is that at least some forms of denaturation (such as pressure-induced) involve penetration of water into the hydrophobic interior. But that picture is challenged in a paper by Santosh Kumar Jha and Jayant Udgaonkar at the Tara Institute of Fundamental Research in Bangalore, at least for the case of denaturant-induced (GdnHCl) unfolding (PNAS 10.1073/pnas.0905744106; paper here). They have used UV circular dichroism measurements on the small plant protein monellin to show that here unfolding seems to involve a dry molten-globule intermediate, reached from the native state in a rather sharp configurational transition.

Also on this topic, Paul Cremer and colleagues at Texas A&M have used FTIR and thermodynamic measurements to probe the notion that urea denatures via direct hydrogen-bonding to the protein surface (L. B. Sagle et al., JACS 131, 9304 (2009); paper here). They challenge that idea, saying that hydrogen-bonding of urea seems to actually promote hydrophobic collapse of a polyamide (PNIPAM, here used as a protein analogue).

Ahmed Zewail and his colleagues have looked at how solvent motion couples to the unfolding of a model protein (melittin) in the presence of a denaturant (trifluoroethanol) (C. M. Othon et al., PNAS 10.1073/pnas.0905967106; paper here). Using time-resolved fluorescence spectroscopy, they see an abrupt change in solvent dynamics at a critical TFE concentration associated with a change in protein structure, in which the tetramers dissociate into loosely bound monomers owing to penetration of TFE into the hydrophobic core. The dissociation of the monomers happens in a distinct second step.

Sapna Sarupria and Shekhar Garde at RPI have studied the compressibility and fluctuations of hydration shells of hydrophobic solutes and proteins using MD simulations (Phys. Rev. Lett. 103, 037803; 2009 – paper here). They say that the compressibility is non-monotonic as a function of solute size, and that it is greater near hydrophobic solutes, relative to the bulk. The latter implies that hydrophobic interactions get weaker as pressure is increased, which may be important for pressure-induced denaturation. This pressure sensitivity is also dependent on the curvature of the solute, being greater for low-curvature surfaces. That might have a role in the pressure dissociation of multi-subunit proteins.

Incidentally, I am preparing a feature article on denaturation for Chemistry World, and would welcome any papers that might be relevant to this.

Padmanabhan Balaram and colleagues at the Indian Institute of Science in Bangalore report a very nice crystal structure of a model peptide containing a hydrophobic channel containing a linear water wire of nine molecules (U. S. Raghavender et al., JACS 10.1021/ja9038906; paper here). Looks like a good model system for studying such structures.

There is a clutch of papers on hydration dynamics of proteins. A painstaking study of femtosecond dynamics in the hydration network of apomyoglobin by Dongping Zhong and colleagues at Ohio State has revealed two distinct classes of water-network relaxation (L. Zhang et al., JACS 10.1021/ja902918p; paper here). One comes from collective hydrogen-bond rearrangements in the water shell, while the other is considerably slower and results from coupled water-protein motions. They are also able to follow changes in these motions in the transition from the native to the molten-globule state. This looks like the kind of careful study that is needed to really figure out what the intimate coupling of protein and water motions entails.

M. Vogel at the Technical University of Darmstadt has used MD simulations to look at how the dynamics of the hydration shells of peptide analogues of structural proteins (elastic and collagen) change with temperature (J. Phys. Chem. B 113, 9386 (2009); paper here). He finds that there is a change from diffusive motion at higher temperatures to jump-like motion on cooling, corresponding to a weak fragile-to-strong crossover of the water dynamics.

And Giorgio Schirò and colleagues at the University of Rome III use dielectric spectroscopy to study the dynamics of myoglobin confined in porous silica at low hydration levels, with only one or two layers of water around the protein (G. Schirò et al., J. Phys. Chem. B 113, 9606 (2009); paper here). They find that confinement has a big effect relative to hydrated myoglobin powder, suppressing the cooperativity of the water motions and the strong coupling to the protein dynamics. All the same, there still seems to be some slaving of protein relaxation in the porous medium to one mode of solvent relaxation.

Dor Ben-Amotz and colleagues at Purdue have seen the spectroscopic signature of dangling OH bonds in the hydration shells of small dissolved nonpolar molecules, similar to those seen at macroscopic water-oil interfaces (P. N. Perera et al., PNAS 10.1073/pnas.0903675106; paper here). And Pier Luigi Silvestrelli at the University of Padova offers further evidence, from first-principles calculations, that hydrophobic groups (here the methyl of methanol) don’t immobilize water molecules, iceberg-like, in the immediate hydration shell, but rather, merely slow down many surrounding molecules (J. Phys. Chem. B 10.1021/jp9044447; paper here).

It’s occasionally and justifiably said that too little attention has been given to polysaccharide hydration, in contrast to proteins. As a result, we know relatively little about glycoprotein hydration. Claudio Margulis and colleagues at Iowa State attempt to redress that imbalance somewhat with a paper looking at the hydration shells of a diverse range of carbohydrates (S. K. Ramadugu et al., J. Phys. Chem. B 10.1021/jp904981v; paper here). I’m not sure I can easily summarize the results, but there looks to be a lot of valuable information here, for example in terms of how water structure and dynamics are affected by branching, size and type of linkage in the polysaccharides.

The behaviour of water in carbon nanotubes continues to intrigue as a model for hydrophobic protein pores. MD simulations by Biswaroop Mukherjee of the Indian Institute of Science and coworkers suggest that jump reorientation of water molecules inside narrow nanotubes involves a switch of which of the two hydrogens are H-bonded to a neighbour, in contrast to such jumps in the bulk which involve H-bonding to a different neighbour (B. Mukherjee et al., J. Phys. Chem. B 10.1021/jp904099f; paper here).

Francesco Mallamace, Gene Stanley and their collaborators have a paper in Nature Physics that describes the appearance of a fractional Stokes-Einstein relation (which provides information about viscosity, connecting the self-diffusion coefficient to temperature and relaxation time) below 290 K in water confined within silica nanopores (2 nm diameter) (Nature Physics 10.1038/nphys1328; paper here). They suggest that this switch marks a crossover to a water structure that is locally more similar to LDA ice – a point at which the proportions of HDA-like and LDA-like configurations starts to change rapidly. It’s an intriguing idea, and there is apparently some indication (I can say no more yet) that the dynamical crossover might be a more general phenomenon for liquids. Some, though, will reasonably wonder whether water within 2-nm silica pores can really be considered representative of the bulk.

Perhaps relevant in this respect, Patrick Huber at Saarland University in Germany and colleagues have studied the dynamics of capillary rise in silica pores 3-5-5 nm across (S. Gruener et al., Phys. Rev. E 79, 067301 (2009); paper here). They find that they can account for the timescales of pore filling according to macroscopic hydrodynamics, so long as they assume the presence of a ‘sticky preadsorbed boundary layer of about two monolayers of water molecules’. In other words, there is dynamical partitioning of the water ‘filling’ into two components.

Masakazu Matsumoto at Nagoya University offers a new picture of the density maximum of water cooled towards freezing (Phys. Rev. Lett. 103, 017801 (2009); paper here). He challenges the common, somewhat arm-waving idea that the decrease in density below 4 C is due to a dominance of LDA-like local configurations. Rather, he says, a proper description of the associated structural changes needs to be broken down in more detail: the anomaly seems to stem from a combination of the change in average hydrogen bond length as a function of temperature (which is monotonic) and the contraction of the HB network due to bond-angle distortion. I’m imagining (it is not made explicit) that this differs from a decline in H-bond-breaking caused by a switch from HDA-like to LDA-like.

Yizhak Marcus at the Hebrew University of Jerusalem uses standard partial molar volumes to calculate the hydration numbers of a range of univalent and divalent ions under ambient conditions (J. Phys. Chem. B 10.1021/jp9027244; paper here). And László Pusztai at the Hungarian Academy of Sciences and colleagues use simulations and neutron/X-ray diffraction to find the hydration numbers of CsCl over a range of concentrations (V. Mile et al., J. Phys. Chem. B 10.1021/jp900092g; paper here). But the numbers don’t agree: Marcus calculates hydration numbers of 1.5-2.5 (depending on the definition) for Cs and 1.4-2.0 for Cl (at 25 C), whereas Pusztai et al. find respective figures of 8-6.5 (for increasing salt concentration) and 5-7. I’m not clear why the numbers are so different. Thanks to Jan Engberts for pointing me to these two papers.

How do specific ions bind to protein surfaces? The answer to this question promises to shed light on the much debated Hofmeister effects on protein aggregation, but there is still no consensus. One common rule of thumb talks of the law of ‘matching water affinities’, whereby cations and anions form ion pairs if they are more or less matched in size. Berk Hess and Nico van der Vegt present simulations which suggest that this simple relationship breaks down for alkali metal cations binding to carboxylate groups on protein surfaces (PNAS 10.1073/pnas.0902904106 – paper here). They argue that the picture is more complicated than such as simple physico-chemical law can express, involving the nature of the hydrated ion complex and the possibility of water-bridged interactions.

The mechanism of fast proton transport in water has been long debated, with the traditional view of Grotthus-like proton hopping along water chains now refined to a picture that tends to invoke intermediates of either the Eigen or Zundel ions (H3O+.3H2O or H5O2+). Rather similar considerations have been applied to the transport on hydroxide ions – are they just a mirror image of proton transport, or do they involve other ionic species such as H3O2-? Andrei Tokmakoff at MIT and his coworkers now present femtosecond pump-probe IR spectroscopic results that they say points to the significant involvement of a Zundel-like transition state in proton transfer in hydroxide solutions, in which a proton is delocalized between a hydroxide ion and a water molecule (S. T. Roberts et al., PNAS 10.1073/pnas.0901571106 – paper here).

Shuxun Cui at Southwest Jiaotong University in Chengdu has published a paper in which he speculates about the prebiotic implications of his recent single-molecule force spectroscopy work (some with Herman Gaub) on the structures of DNA in water and non-aqueous media (see for example JACS 128, 6636 (2006) and JACS 129, 14710 (2007)). He argues that double-stranded DNA can be seen as an adaptation to an aqueous environment (S. Cui, IUBMB Life 61, 860 (2009) – paper here). I have the strong sense that this, rather than Lawrence Henderson’s ‘fitness of the environment’, is the right way round to be examining this question.