Stabilizing and destabilizing proteins

How do osmolytes stabilize proteins against denaturation? Using mechanically induced unfolding by means of atomic-force microscopy of protein I27 (in fact 8 repeat units in a polyprotein), Julio Fernández and colleagues at Chicago find that osmolytes can act as ‘solvent bridges’ during the unfolding transition, pinning together hydrogen-bonding sites on the polypeptide backbone (L. Dougan et al., PNAS 108, 9759; 2011 – paper here). They found in earlier studies that water molecules generally act in this capacity. They now investigate this process in the presence of glycerol, which is known to enhance protein stability, and related solutes. They find that while glycerol, ethylene glycol and propylene glycol all enhance the mechanical stability, larger hydrogen-bondong osmolytes such as sucrose and sorbitol do so to a far lesser extent – MD simulations suggest that the latter are unable to penetrate the folded structure and act as stabilizing bridges, and any stabilizing influence they exert must be indirect.

The effects of methanol on protein conformational stability are even more complex, to judge from the NMR and MD results of Christian Hilty of Texas A&M and coworkers (S. Hwang et al., J. Phys. Chem. B 115, 6653; 2011 – paper here). They report that the alcohol stabilizes the secondary structure by strengthening hydrogen-bonding interactions in the backbone (a consequence of partially eliminating the water molecules that compete with these interactions), while also weakening hydrophobic interactions and thus swelling and loosening the folded structure overall. So whether methanol tightens or loosens the structure overall is a complex balance that depends on the sequence and initial structure of the protein.

Urea and guanidinium both help to denature the model protein the Trp-cage, according to the experiments and simulations of Pavel Jungwirth in Prague and colleagues (J. Heyda et al., J. Phys. Chem. B 10.1021/jp200790h – paper here). Although these two small molecules are chemically quite different, it appears that their denaturing mechanisms here are essentially the same, involving first the destabilizing displacement (positional exchange) of two proline residues within the hydrophobic core, followed by a gradual unravelling of secondary structural elements.

The conformational stability of proteins is also influenced by ions: Hofmeister ‘salting out’ agents such as the strongly solvated, high-charge carbonate and sulphate ions also stabilize the folded state, whereas weakly solvated low-charge ions such as bromide and iodide promote denaturation. This tendency overlaps with the classical denaturation properties of complex ions such as guanidinium (Gdm+). Christopher Dempsey at Bristol and colleagues have studied these effects for the cases of the sulphates and chlorides of guanidinium and tetrapropylammonium (TPA+) (C. Dempsey et al., JACS 133, 7300; 2011 – paper here). They find that the question of conformational stabilization is subtle. TPA+, like Gdm+, perturbs the stability of some proteins (specifically the tryptophan zipper trizip, a beta-hairpin peptide), but stabilizes the alpha-helices of alahel peptides, apparently because TPA+ cannot compete effectively for hydrogen bonds in H-bond-stabilized conformations. Moreover, the cation effects may be modified by the anion: sulphate counteracts the denaturing effects of Gdm+ on trizip, but has no effect on the influence of TPA+ in that case, because Gdm+ but not TPA+ forms ion pairs with sulphate. The emerging picture is thus one in which the Hofmeister-like effects of ions must be understood in the light of a detailed consideration of (i) ion hydration; (ii) anion-cation interactions; and (iii) direct ion-protein interactions.

More on Hofmeister from Corinne Gibb and Bruce Gibb of the University of New Orleans, who offer a new perspective: namely, that so-called chaotropic anions in fact display bind preferentially to concave hydrophobic surfaces, thus effectively weakening hydrophobic attraction and promoting ‘salting in’ (JACS 133, 7344; 2011 – paper here). They reach this conclusion by looking at the thermodynamics of ion effects in the binding of adamantane carboxylic acid within the deep hydrophobic cavity of a cavitand – an interaction perturbed by anions binding to the cavity. Sure, the extension to proteins is purely by analogy, but it’s an intriguing take on the old problem. Meanwhile, Oleg Krasilnikov and colleague at the Federal University of Pernambuco in Brazil have considered how ions affect molecular interactions in nanopores, and find that these too seem to display a Hofmeister-like sequence of activities (C. G. Rodrigues et al., Biophys. J. 100, 2929; 2011 – paper here). They look at how simple ions alter the rate constant for the interaction of poly(ethylene glycol) with the protein pore alpha-hemolysin, and see a change consistent with the Hofmeister series for halide anions. They suggest that this results from a competition for hydration water between the ions and other solutes within the pore.

Protein aggregation is commonly suppressed by the addition of surfactants and sugars or polyols. But arginine hydrochloride has also been found to possess this capability, and unlike some conventional aggregation-suppressors it does so by reducing protein-protein interactions without seeming to affect the stability of the folded conformation. This behaviour hasn’t been fully explained, although Bernhardt Trout and colleagues at MIT have proposed that at least part of the mechanism might be non-specific entropic effects due to the exclusion of arginine from the gap between two proteins as they come together (B. M. Baynes et al., Biochemistry 44, 4919; 2005). They now refine this picture by looking at the influence of the anionic counterion, showing that these display the usual Hofmeister progression for aggregation suppression (C. P. Schneider et al., J. Phys. Chem. B 10.1021/jp111920y – paper here). The protonated arginine contains a guanidinium group, again potentially making the connection to denaturant activity – but the relevant behaviour here seems in fact to be the ability of the arginine ions to self-associate in stacks. Thus, while arginine can, like Gdm+, bind directly to the protein surface, the self-association weakens this interaction so that it does not cause denaturation yet still weakens protein-protein interactions. Schneider et al. elucidate this process further by looking, both experimentally and computationally, at the anion effects. Some anions, such as sulphate, phosphate and citrate, can enhance the cationic clustering by forming multiple hydrogen bonds, creating larger clusters and thus stronger exclusion effects on aggregation – and perhaps also slowing protein diffusion via an enhancement of solvent viscosity.

Understanding how bacteria void toxic substances from the cell interior could have a profound impact on our ability to combat antibiotic resistance. E. coli have a multi-drug efflux pump called AcrAB-TolC, in which the AcrB protein in the inner membrane binds drugs non-specifically and pumps them to the TolC exit duct. Attilio Vargiu of the University of Cagliari and colleagues have taken a close look at how this pump works, with a particular focus on the role of water molecules in carrying the extruded substrate along (R. Schulz et al., J. Phys. Chem. B 10.1021/jp200996x – paper here). The protein has several small holes that allow water molecules to enter and flow in a directional manner. This water acts as a lubricant and transport medium for the drug, but also flattens out the electrostatic profile in the channel that might otherwise cause the drugs to get stuck (perhaps by hydrogen bonding), and thus it contributes to the polyspecificity of the mechanism.

The role of water-mediated interactions in protein-substrate binding and associated drug design has been given a fair bit of attention, but W. David Wilson of Georgia State University and colleagues show that such things may be relevant to small-molecule DNA-binding agents too. They look at the binding of the synthetic molecule DB921 into the AT-rich minor groove of DNA, an interaction that might be useful for the disruption of parasite mitochondria (Y. Liu et al., JACS 10.1021/ja202006u – paper here). The binding is mediated by a water molecule, and to better understand how this works the researchers look at the effect on the structure, kinetics and thermodynamics of binding of introducing a host of modifications to DB291. This information, in particular the characteristics responsible for the water-mediated interaction, could be valuable for designing new agents that bind strongly in the minor groove in a sequence-specific manner.

Combining vibrational sum-frequency measurements with simulations containing three-body terms, James Skinner and colleagues at the University of Wisconsin say that the liquid-vapour interface of water shows no evidence of ‘enhanced’ molecular structuring such as ice-like ordering (P. A. Pieniazak et al., JACS 10.1021/ja2026695 – paper here).

The charge and pH of this interface have recently become contentious issues. Sylvie Roke at the MPI for Metals Research in Stuttgart and colleagues now offer a profile of the oil-water interface, using zeta-potential and sum-frequency scattering measurements alongside MD simulations (R. Vácha et al., JACS 10.1021/ja202081x – paper here). They say that the water orientations at the surface are like those at a negatively charged surface, even though there is no hydroxide absorption to make it so. There is nonetheless a surface charge, which comes instead from a disturbance in the balance of hydrogen-bond donors and acceptors at the interface.

The notion that the hydration water of proteins is dynamically coupled to the protein itself receives more support from a study by Nguyen Quang Vinh and colleagues at UCSB, who have used terahertz spectroscopy to look at the large-scale collective vibrations of lysozyme (N. Q. Vinh et al., JACS 133, 8942; 2011 – paper here). They find that the protein is surrounded by 150-180 water molecules (a sub-monolayer) that, in the authors’ words, ‘in terms of their picosecond dynamics behave as if they are an integral part of the protein’. THz spectroscopy seems to be emerging as a nigh-incomparable technique for probing these long-ranged collective motions.

The dynamics of hydration water are also studied by Cesare Cametti at the University of Rome ‘La Sapienza’ and colleagues, using dielectic spectroscopy (C. Cametti et al., J. Phys. Chem. B 10.1021/jp2019389 – paper here). They find that the dielectric relaxation of the lysozyme hydration sphere is bimodal at high concentrations, corresponding to tightly and loosely bound waters, although monomodal in dilute solution.

The nature of the protein dynamical transition at 200-220 K shows no sign of being resolved. Salvatore Magazù and colleagues at the University of Messina now throw a cat among the pigeons by suggesting that there is no such transition at all: it is an artefact caused by the coincidence of the system’s relaxation time with the instrumental resolution (S. Magazù et al., J. Phys. Chem. B 115, 7736; 2011 – paper here). Nonetheless, they say – can life really be this complicated? – there is a crossover of some sort at 220 K, at least for the case of lysozyme considered here, for this marks a change from Arrhenius to super-Arrhenius behaviour in the coupled hydration-water/protein motions. I think what this implies – it is a little unclear – is that there is no intrinsic, qualitative change in the protein dynamics at the ‘dynamical transition’, but rather, a change due to the coupling of these to the hydration water dynamics. I could be wrong.

Water rotational dynamics are slowed down around many small solutes, both hydrophilic and hydrophobic. So it stands to reason that the same should happen for larger solutes, such as proteins. And it does – but Ana Vila Verde and R. Kramer Campen at the FOM Institute in Amsterdam suggest that the latter is not necessarily just a straightforward extension of the former (J. Phys. Chem. B 10.1021/jp112178c – paper here). Their simulations of water dynamics around the disaccharides kojibiose and trehalose show that the sheer size of these solutes, relative to smaller ones, induces additional mechanisms of water retardation as a result of topological constraints on water motions. Thus, one can’t for example imagine that the hydration of a free amino acid is the same as that when the amino acid represents a peptide residue.

Bear that in mind, perhaps, in considering the hydration structure of glycine as deduced from ab initio calculations by Bo Liu at Henan University and colleagues. They build up the hydration shell molecule by molecule from a gas-phase picture (Y. Yao et al., J. Phys. Chem. B 115, 6213; 2011 – paper here).

Salt bridges play a role in stabilizing the glycosyl hydrolase (an enzyme with potentially important industrial applications) of the hyperthermophile Rhodothermus marinus (L. Bleicher et al., J. Phys. Chem. B 115, 7940; 2011 – paper here). But perhaps surprisingly, the enzyme also contains salt bridges that seem to be destabilizing: located in the hydrophobic core, where they might facilitate the permeation of water.

C. Preston Moon and Karen Fleming at Johns Hopkins have a neat idea for developing a hydrophobicity scale for amino acids based on their energy of transfer from water to within a phospholipid bilayer, thus relating the measure directly to the driving forces for the assembly and stabilization of membrane proteins (PNAS 108, 10174; 2011 – paper here). Their thermodynamics measurements show some differences from the predictions of simulations, especially for the translocation of arginines – which makes the results relevant to voltage-sensitive ion channel gating mechanisms, since these involve the movement of arginines into the hydrophobic interior of the membrane.

How homogeneous are concentrated aqueous solutions? This question has been studied for methanol, which seems to aggregate to some extent in water; now Lorna Dougan at the University of Leeds and colleagues study the case of glycerol using neutron scattering, motivated by the relevance to cryoprotection (J. J. Towey et al., J. Phys. Chem. B 115, 7799; 2011 – paper here). They find that glycerol-glycerol hydrogen-bonding in the pure liquid is scarcely affected by the addition of water, while the water-water bonding is highly disrupted. In effect, the waters become isolated from one another, binding preferentially to glycerols. Thus, it seems likely that glycerol could act as a cryoprotectant by keeping water molecules apart and preventing for the formation of an ice network.

Water can penetrate the hydrophobic interior of carbon nanotubes, a fact that is being investigated for potential desalination technologies among other things. William Goddard at Caltech and colleagues take a look at what drives the filling process for different tube diameters (T. A. Pascal et al., PNAS pnas.1108073108 – paper here). For CNTs between 0.8 and 2.7 nm, the interior water phase is always more stable than the bulk, but for several different reasons. For nanotubes thinner than 1 nm, the water phase is gas-like, with an entropic driving force. For nanotubes of 1.1-1.2 nm the encapsulated phase is ice-like and enthalpy-stabilized. For nanotubes wider than 1.4 nm the interior phase is liquid-like, but stabilized by increased translational energy. The overall message is sobering in showing how very fine adjustments to the hydrogen-bonded network and the balance of interactions with the surrounding environment can create quite different phase behaviour and thermodynamic driving forces in confined situations even for very small differences in dimensions.

A new method for rapidly calculating solvation energies in water is presented by Jianzhong Wu and coworkers at the University of California at Riverside (S. Zhao et al., J. Phys. Chem. B 10.1021/jp201949k – paper here), which combines DFT with MD simulations. The researchers have so far tested it for simple ions, for which it works well. Meanwhile, Merchant and Dilip Asthagiri at JHU have a preprint (arxiv 1106.0448 – paper here) in which they examine the range of ion-specific effects in water and conclude that, at least for sodium, potassium, chloride and fluoride, these extend no more than about 4 Å, so not much more than the size of a single water molecule.

This hasn’t yet exhausted my list of papers: still to come (soon) are developments on the putative liquid-liquid transition of water and on proton transport in bacteriorhodopsin…