Changes in solvation play a central role in the thermodynamics of non-covalent interactions in solution, especially in water, yet there are relatively few techniques available to probe this unambiguously. Experimental studies of the thermodynamics of biomolecular interactions in water have exposed two significant empirical observations. The first, well known from the very earliest applications of microcalorimetry, is that processes such as protein folding, ligand binding, and protein–protein association almost always occur with a decrease in overall heat capacity of the system (negative ΔCp). This results in a strong temperature dependence of the enthalpy of interaction that has, historically, been usually attributed to solvation changes, though more generally it has been shown to be an inevitable consequence of processes involving the cooperative interaction of multiple weak interactions. More recently using pressure perturbation calorimetry (PPC), we have shown that such interactions in the same systems also occur with significant decreases in molar thermal expansibility (negative ΔE°) that can be related to the loss of solvation during complexation. The apparently strong correlation between ΔCp and ΔE° potentially leads to a generic picture of the thermodynamics of macromolecular interactions in water in which both solvation and conformational fluctuation play a much more prominent role than has been hitherto supposed.

Naturally occurring foam constituent and surfactant proteins with intriguing structures and functions are now being identified from a variety of biological sources. The ranaspumins from tropical frog foam nests comprise a range of proteins with a mixture of surfactant, carbohydrate binding and antimicrobial activities that together provide a stable, biocompatible, protective foam environment for developing eggs and embryos. Ranasmurfin, a blue protein from a different species of frog, displays a novel structure with a unique chromophoric crosslink. Latherin, primarily from horse sweat, but with similarities to salivary, oral and upper respiratory tract proteins, illustrates several potential roles for surfactant proteins in mammalian systems. These proteins, together with the previously discovered hydrophobins of fungi, throw new light on biomolecular processes at air–water and other interfaces. This review provides a perspective on these recent findings, focussing on structure and biophysical properties.

Pressure perturbation calorimetry measurements on a range of cyclodextrin−adamantane, protein−ligand (lysozyme−(GlcNac)3 and ribonuclease−2′CMP) and protein−protein (cytochrome c peroxidase−pseudoazurin) complexes in aqueous solution show consistent reductions in thermal expansibilities compared to the uncomplexed molecules. Thermodynamic data for binding, obtained by titration calorimetry, are also reported. Changes in molar expansibilities can be related to the decrease in solvation during complexation. Although reasonable estimates for numbers of displaced water molecules may be obtained in the case of rigid cyclodextrin−adamantane complexes, protein expansibility data are less easily reconciled. Comparison of data from this wide range of systems indicates that effects are not simply related to changes in solvent-accessible surface area, but may also involve changes in macromolecular dynamics and flexibility. This adds to the growing consensus that understanding thermodynamic parameters associated with noncovalent interactions requires consideration of changes in internal macromolecular fluctuations and dynamics that may not be related to surface area-related solvation effects alone.

Protein unfolding in aqueous solution is usually accompanied by an increase in heat capacity (ΔCp), and this has long been regarded as somewhat anomalous. However, neither the absolute heat capacities (Cp) of folded globular proteins nor the heat capacity increments upon unfolding (ΔCp) are unusual in comparison to values observed for order−disorder (melting) transitions in other organic substances. The consequences for protein stability, including cold denaturation, enthalpy−entropy compensation, and the temperature of maximum stability, may seem counterintuitive but should not be unexpected. Nevertheless, while perhaps not so anomalous as once thought, quantitative interpretation of protein heat capacity and related effects remains a theoretical challenge.

Modern techniques in microcalorimetry allow us to measure directly the heat changes and associated thermodynamics for biomolecular processes in aqueous solution at reasonable concentrations. All these processes involve changes in solvation/hydration, and it is natural to assume that the heats for these processes should reflect, in some way, such changes in solvation. However, the interpretation of data is still somewhat ambiguous, since different non-covalent interactions may have similar thermodynamic signatures, and analysis is frustrated by large entropy–enthalpy compensation effects. Changes in heat capacity (ΔCp) have been related to changes in hydrophobic hydration and non-polar accessible surface areas, but more recent empirical and theoretical work has shown how this need not always be the case. Entropy–enthalpy compensation is a natural consequence of finite ΔCp values and, more generally, can arise as a result of quantum confinement effects, multiple weak interactions, and limited free energy windows, giving rise to thermodynamic homeostasis that may be of evolutionary and functional advantage. The new technique of pressure perturbation calorimetry (PPC) has enormous potential here as a means of probing solvation-related volumetric changes in biomolecules at modest pressures, as illustrated with preliminary data for a simple protein-inhibitor complex.

Ranaspumin-2 (Rsn-2) is a monomeric, 11 kDa surfactant protein identified as one of the major foam nest components of the túngara frog (Engystomops pustulosus), with an amino acid sequence unlike any other protein described so far. We report here on its structure in solution as determined by high-resolution NMR analysis, together with investigations of its conformation and packing at the air-water interface using a combination of infrared and neutron reflectivity techniques. Despite the lack of any significant sequence similarity, Rsn-2 in solution adopts a compact globular fold characteristic of the cystatin family, comprising a single helix over a four-stranded sheet, in a motif not previously associated with surfactant activity. The NMR structure of Rsn-2 shows no obvious amphiphilicity that might be anticipated for a surfactant protein. This suggests that it must undergo a significant conformational change when incorporated into the air-water interface that may involve a hinge-bending, clamshell opening of the separate helix and sheet segments to expose hydrophobic faces to air while maintaining the highly polar surfaces in contact with the underlying water layer. This model is supported by direct observation of the relative orientations of secondary structure elements at the interface by infrared reflection absorption spectroscopy, and by protein packing densities determined from neutron reflectivity profiles.