Thursday, April 25, 2013

Hydration and thermostability

What makes thermophilic proteins stable? And in particular, does hydration play a role? Those questions are examined by Fabio Sterpone of the Université Paris Diderot and colleagues (O. Rahaman et al., Phys. Chem. Chem. Phys. 15, 3570; 2013 – paper here). They study two homologous proteins, one from E. coli and the other from the thermophile Sulfolobus solfataricus, using MD simulations. The average water dynamics at the protein surface is the same in both cases, despite their different amino acid compositions, being slowed by a factor of 3-5 relative to the bulk. The authors conclude that this slowdown is primarily a geometric effect due to excluded volume, which explains why the protein sequence has little influence. This doesn’t exactly answer the initial question about thermostability – but it does suggest that there is nothing special about thermophilic proteins in terms of their hydration.

More on dewetting as a mechanism for protein folding and stability. Ruhong Zhou, Robert Matthews and their colleagues at Columbia have simulated a TIM barrel protein and found drying inside clusters of hydrophobic residues, signified by strong fluctuations in water density (P. Das et al., JACS 135, 1882; 2013 – paper here). In particular, a hydrophobic cluster (ILV) at the N-terminus shows drying that is weakened or suppressed by substituting some of the hydrophobic amino acids for less hydrophobic ones. A cluster at the C-terminus, meanwhile, seems to experience only partial drying that is unaffected by such substitutions. Experiments on the structure and stability of wild-type and mutant versions seem to back up these conclusions. The authors note that ILV clusters are common in several other protein motifs too.

Huib Bakker and colleagues at the FOM Institute in Amsterdam have used Förster resonant energy transfer between water OH stretches to find out where the water hydrating lipid membranes actually is (L. Piatkowski et al., J. Phys. Chem. B 117, 1367; 2013 – paper here). Somewhat surprisingly, they find that, even at rather low hydration levels, the water molecules are not evenly distributed among the lipid head groups, but form nanoscale clusters with an average intermolecular distance of 3.4 Å.

Our picture of the water surface continues to evolve. Is the water here structurally and dynamically different from that in the bulk? That idea is supported by experiments by James Skinner and colleagues at Wisconsin in simulations using a new three-body water potential (Y. Ni et al., PNAS 110, 1992; 2013 – paper here). They calculate that the hydrogen-bond switching dynamics are retarded at the water surface by a factor of about 3, although the rotational dynamics are actually a little faster. They say that vibrational 2D sum-frequency generation spectroscopy should be a good experimental method for investigating these dynamics, and calculate what the spectra should look like.

To make optimal use of plant cellulose as a feedstock for biofuels such as ethanol, it’s necessary to get it into aqueous solution. But cellulose is hard to solvate, which is why it is important to understand its hydration structure. Sylvia McLean at Oxford and colleagues have used neutron diffraction to look at the aqueous solvation of the disaccharide cellobiose, and find that there is (as has been suggested) a hydrogen bond across the glycosidic bond linking the two sugars (W. B. O’Dell et al., PLoS ONE 7, e45311; 2012 – paper here). There is competition from water molecules for the oxygen acceptor in this bond, however, with average occupancy of 50% for both water and the intramolecular OH donor.

More evidence that a hydrogen-bonded cluster of water molecules at the catalytic site plays a crucial role on photosynthesis: Bridgette Barry and colleagues at Georgia Tech use EPR to look at the rate of reaction of an intermediate neutral radical in the proton-coupled electron transfer that leads to oxygen evolution at the reaction centre of photosystem II, which contains a catalytic Mn4CaO5 cluster (J. M. Keough et al., J. Phys. Chem. B 117, 1296; 2013 – paper here). They find that this network is rearranged during the transition between the S0 and S2 states of the catalytic cycle, and that ammonia slows oxygen evolution because it disrupts the network by displacing water.

Suzi Jarvis and colleagues at University College Dublin have been using scanning probe microscopy for some time to probe hydration forces, and in their latest contribution they use a technique called frequency modulation AFM to look at hydration forces at the interface of mica and an electrolyte (J. I. Kilpatrick et al., JACS 135, 2628; 2013 – paper here). By ‘hydration force” here they mean the monotonically decaying force with a characteristic length of a few Å, without regard to its precise origin. They find that, relative to pure water, ions introduce or accentuate oscillations in the force as a function of distance due to the formation of distinct hydration layers. They point out that there are implications for obtaining atomic-resolution AFM images in aqueous saline solution.

Uzi Landman, Gary Schuster and colleagues at Georgia Tech offer a fascinating insight into the role of hydration water around DNA in the oxidation and consequent mutation of A/T-rich regions, which my be significant in the early stages of carcinogenesis due to stalling of replication (R. N. Barnett et al., JACS 135, 3904; 2013 – paper here). Their experiments and simulations indicate that oxidation of adenine leads to proton-coupled electron transfer to thymine, mediated by a water wire. This process can explain why nearly all the mutation due to such an oxidation event happens at thymine.

It’s sobering to realise that even now the reasons for the anomalous behaviour of water’s thermodynamic response functions, such as the divergence in the heat capacity and compressibility at low temperature, are still unknown. Francesco Mallamarce, Carmelo Corsaro and Gene Stanley discuss this issue with reference to experimental data on the power spectrum of sound velocity (F. Mallamarce et al., PNAS 110, 4899; 2013 – paper here). They conclude that the anomalies are associated with a structural transformation due to the appearance of an extended hydrogen-bonding network, which gives rise to viscoelastic behaviour in the liquid.

Also on bulk water, what happens to water’s structure above its critical point has been studied using X-ray Raman spectroscopy by Christoph Sahle of the Technical University of Dortmund and colleagues (C. Sahle et al., PNAS 110, 6301; 2013 – paper here). They find that, as one might expect, distortions of the hydrogen bonds are significant above the critical point, and the average coordination of each molecule decreases to just 0.6 at 600 oC and 134 MPa.

Friday, April 12, 2013

How antifreeze proteins work

A relatively short catch-up, with much more to come…

Martina Havenith at Bochum and her colleagues have extended their previous work on the mechanism of antifreeze proteins. Previously they looked at an antifreeze glycoprotein and found, using terahertz spectroscopy, that ice-binding by the protein seems to involve a long-range retardation of water H-bond dynamics, extending up to 2 nm from the molecular surface (S. Ebbinghaus et al., JACS 132, 12210; 2010). Now they find a similar effect operating for the antifreeze protein (AFP) of the fire-coloured beetle D. canadensis, a hyperactive insect (K. Meister et al., PNAS 110, 1617; 2013 – paper here). This contrasts, however, with the mechanism of another class of AFP, called wfAFP-1, which seems to operate only by short-ranged water binding to surface OH groups (S. Ebbinghaus et al., Biophys. J. 103, L20; 2012). As the authors say, “Nature is probably more inventive than initially thought and makes use of short- and long-range water perturbation to varying degrees in different classes of AFPs”.

Meanwhile, Ido Braslavsky of Ohio University and colleagues have used microfluidic methods to study the effects of AFPs on ice nucleation, and find that the growth of ice crystals is inhibited by the irreversible surface binding of the proteins (Y. Celik et al., PNAS 110, 1309; 2013 – paper here). These results help to rule out suggestions that direct binding of the AFPs to ice is not necessary to their mode of action.

More cases of water assisting receptor-substrate recognition and catalytic activity. First, Stephen Neidle at UCL and colleagues find that a cluster of 11 water molecules in an AT region of the minor groove of DNA seems to support the binding of three different small-molecule ligands (D.-G. Wei et al., JACS 135, 1369; 2013 – paper here). This cluster stabilizes the ligand by hydrogen bonding, and is also linked to (but distinct from) the well-known spine of hydration in B-DNA. Slight differences in binding mode with this cluster seem to account for the differences in binding affinity of the ligands: in other words, it is the water network that is calling the shots.

Second, Xiaoqing Wang and Hajime Hirao at Nanyang Technological University in Singapore say that the catalysis of myo-inositol monophosphatase (IMPase, a potential target for lithium treatment of bipolar disorder) is dependent on two bound water molecules (J. Phys. Chem. B 117, 833; 2012 – paper here). One provides the hydroxide ion that attacks the bound substrate. The other, coordinated to a magnesium ion, facilitates proton transfer leading to the product.

There seems to be an increasing perception that understanding the collective vibrations of proteins – their softness or rigidity – could offer insights into their enzymatic activity. Sow-Hsin Chen at MIT and coworkers support that view with an X-ray scattering study of the collective modes of hydrated lysozyme (Z. Wang et al., J. Phys. Chem. B 117, 1186; 2013 – paper here). They find that at low hydration levels both the collective ‘soft’ phonon modes and the enzymatic activity are much weaker or absent, suggesting a causal relationship: an indicator of the now familiar plasticizing effect of hydration.

C. Cametti at “La Sapienza” University of Rome and colleagues consider another aspect of protein hydration: how high concentrations of the protein (again lysozyme) can lead to clustering and a consequent decrease in average hydration number (C. Cametti et al., J. Phys. Chem. B 117, 104; 2013 – paper here). Their measurements of the dielectric spectra from 500 MHz to 50 GHz, which probe orientational relaxation, are consistent with this hypothesis of clustering into small aggregates at concentrations above about 100 mg/mL, which was first proposed by Stradner et al. (Nature 432, 492; 2004).

There is now some debate about whether the proposed two metastable liquid phases and associated critical point claimed on the basis of some simulations is real or not. That has been disputed by David Limmer and David Chandler at Berkeley, who extend their previous negative results using several different water potentials in a new preprint. I have written a commentary on the issue here.