Metastable Mesoscopic Phases in Concentrated Protein Solutions

The Gibbs's definition of a phase assumes completely homogeneous composition, with fluctuations bringing about local variations of less than a few percent. We apply light scattering, atomic force microscopy, and other techniques to demonstrate that even solutions of a single protein of moderate concentration do not comply with Gibbs's definition of phase. In such solutions clusters of sizes from several tens to several hundred nanometers exist and have limited lifetimes. These clusters have a higher free energy than the protein solution, and their lifetime is determined by the barrier for their decay. The clusters affect the viscous and viscoelastic behavior of the solution and are an essential part of potential condensation and aggregation pathways. Since the clusters are observed in solutions of single proteins, they indicate that the proteins have an intrinsic propensity to form mesoscopic structures, which is probably utilized in the formation of the protein complexes in the cytosol. Cluster theories developed for colloid systems appear inapplicable to proteins due to the high level of implied Coulomb repulsion. The experimental evidence on the clusters suggests that their sizes are determined by the kinetics of growth and decay, and not by thermodynamics. A microscopic theory to account for stabilizing and destabilizing factors involving protein molecules and solvent inside the clusters has not yet been developed.