Unraveling the hydrophobic effect, one molecule at a time

Hydrophobicity operates over many scales, from the demixing of oil and water at the macroscopic scale to the folding of proteins in water at the molecular scale. The physics governing hydrophobicity at the two length scales are, however, fundamentally different (1). The hydration of large solutes is governed by surface tension, which favors lower surface area and causes oil drops to coalesce. The surface tension decreases monotonically with increasing temperature, and so does the driving force for coalescence. In contrast, at the microscopic scale, hydrophobic effects vary nonmonotonically, typically becoming stronger and reaching a maximum before decreasing with increasing temperature (e.g., folded proteins can be denatured both by heating and cooling, which implies a maximum in stability as a function of temperature). Theory and simulations predict that the crossover from the molecular to the macroscopic regime occurs at a length scale of the order of 1 nm (1). Because the relevant length scales in proteins range from subnanometer (for side chains exposed in their unfolded states) to several nanometers (in their folded states), understanding of hydration in the crossover region is important for estimating the hydrophobic driving forces in protein folding. Experimental measurements on hydrophobicity in this region have been elusive until now. In PNAS, Li and Walker (2) use single-molecule force spectroscopy of hydrophobic polymers to provide an experimental window into the crossover region.

How Solutes Affect Water Structure

The length-scale dependence of hydrophobicity is, indeed, one of its most fascinating aspects. The difference in the physics of hydration of small and large solutes arises from the different manner in which they affect the structure of water (1, 3–8). Water molecules participate in a locally tetrahedral hydrogen bond network. Thermal fluctuations of that network can accommodate a small hydrophobic solute (9) without sacrificing hydrogen bonds. Such accommodation, however, comes with a significant entropic penalty, reflecting restricted configurations of water molecules in the solute's hydration shell. When small solutes associate, some of these entropically restricted water molecules are released, thereby increasing the water entropy. As a result, the driving force for molecular assembly increases with temperature at ambient conditions before reaching a maximum at higher temperatures. In contrast, a large solute cannot be accommodated in the tetrahedral water network, which results in broken hydrogen bonds, much like at the vapor–liquid interface (1, 3). Hence, interfacial physics (i.e., surface tension) governs the solvation and association of macroscopic solutes.

Pulling Hydrophobic Polymers

Despite significant theoretical and simulation work in this area, experiments have been missing from the picture. A major

Li and Walker succeed in narrowing the gap between theory/simulations and experiments.

hurdle in experimentally probing nanoscale hydrophobic solutes is that they are essentially insoluble in water—solubility of alkanes decreases exponentially with carbon number, falling below an equilibrium mole fraction of about 10⁻¹⁰ for alkanes longer than dodecane (10). Li and Walker's study (2) bypasses this hurdle by using single-molecule force spectroscopy. Although force spectroscopy is now used routinely to study conformational transitions of proteins (11), this innovative work uses it to explore hydrophobicity in the crossover region.

Li and Walker (2) deposit hydrophobic polymer molecules (e.g., polystyrene) on a silicon surface, where the polymers bead up into globules in the presence of water. They use a fine tip of an atomic force microscope to pick up a single polymer molecule and pull it, unraveling the globule one monomer at a time (2). The force required to pull the polymer contains information about the free energy of hydrating the exposed monomers. Repeating the experiments thousands of times and at different temperatures enables them (2) to reliably obtain the variation of free energy with temperature.

Thermodynamic Signatures of the Crossover

For polystyrene, with a monomer size of 7.2 Å, Li and Walker (2) find that the free energy of hydration increases with temperature in the 25–80 °C range, indicating a negative entropy of hydration. This behavior is similar to that of small solutes in water [e.g., the hydration free energy of methane (size ≈ 3.5 Å) also increases with temperature, displaying a maximum at about 150 °C] (12). Thus, although the extended polymer is long, the length scale relevant to its hydration is actually small and determined by the width of a monomer.

Interestingly, for polymers with larger monomers (size = 9.5 and 11.4 Å), although the free energy initially increases with temperature, it displays a maximum within the experimental range. At the free energy maximum, the hydration entropy is zero (i.e., goes from negative to positive). Importantly, the location of the maximum shifts to lower temperatures with increasing monomer size (to 55 °C and 48 °C, respectively). As the size of the solute increases, it becomes harder for water to hydrogen bond around it. Correspondingly, the solvation entropy goes from negative to positive at lower temperatures. For very large solutes, the entropy is expected to be positive, even at ambient conditions, because hydration is governed by interfacial physics.

At a given temperature, the solute size at which entropy crosses from negative to positive values can be used as a definition of the crossover length scale (13). These results suggest that the crossover length scale increases from 3.5 to 9.5 to 11.4 Å as the temperature decreases from 150 °C to 55 °C to 48 °C, respectively. Thus, Li and Walker (2) not only validate theoretical predictions (4, 5) of temperature-dependent hydrophobic hydration, but also provide experimental confirmation that the crossover length scale is of the order of 1 nm near ambient conditions.

Li and Walker's study (2) also shows that the cost to hydrate monomers when they are part of the polymer is smaller than the cost for hydrating them independently. As part of the chain, the monomers are stabilized by hydrophobic interactions with adjacent monomers, even in the extended states. Because the driving force for the collapse of large chains is dominated by the free energy of the extended state, their results caution that estimating the driving force using data from small-molecule hydration alone will likely lead to a significant overestimate.

Simulations of Polymer Collapse

Hydrophobic homopolymers have served as a model system for simulation and theoretical studies of collapse, because they capture the essential physics of hydrophobicity and chain entropy without being encumbered by other specific interactions in proteins. Athawale et al. (14) showed that the thermodynamics of polymer unfolding display protein-like parabolic temperature dependence, with signatures of both heat and cold denaturation. Zangi et al. (15) showed that urea's unfolding action results primarily from direct attractive (enthalpic) interactions with hydrophobic groups on the polymer. And, Miller et al. (16) identified the rate-limiting step in hydrophobic collapse to be the formation of a critical hydrophobic nucleus. Extensions of the work by Li and Walker (2) can potentially make direct connections with these simulation studies (10, 14–16).

What Does the Future Hold?

Understanding hydrophobicity has emerged as a multidimensional challenge. Hydrophobic interactions depend on temperature, pressure, solute size and shape, type, and concentration of additives as well as proximity to interfaces (17). Recent work has highlighted the role of interfaces in modulating assembly. The soft vapor-liquid like nature of an extended hydrophobic interface is predicted to weaken the forces of assembly and also eliminate kinetic barriers in its vicinity (13). Suitable modifications of the experiments by Li and Walker (2) to pull the polymers parallel to the surface (as opposed to perpendicular to it) have the potential to test those interesting predictions at interfaces with diverse chemistries.

As one moves from model solutes to complex realistic systems, new challenges emerge. For example, how does one characterize the hydrophobicity/philicity of patches on the chemically heterogeneous, rugged, and nanoscale interfaces of proteins (18)? Theory and simulations suggest that the answer lies in understanding how water responds to the chemical and topographical context presented by the protein surface (17, 19–22). Specifically, applications of specialized sampling techniques have revealed that it is not the local water density but the rare water density fluctuations (23) or sensitivity of water density to perturbations (1) that enables robust nanoscale characterization of the hydrophobicity of the underlying surface. Other molecular measures of surface hydrophobicity that have been identified include the propensity of a hydrophobic probe to bind to the surface (13, 24) as well as static and dynamic correlations (24–26) in water density near the surface. Experimental measurements of these molecular measures of hydrophobicity are challenging and will require creative methods such as the ones used by Li and Walker (2). By enabling thermodynamic measurements of hydrophobic solutes in the crossover region, Li and Walker (2) succeed in narrowing the gap between theory/simulations and experiments, which is a promising development for exploring the many facets of the hydrophobic effect.