No abstract

Metastable clusters of mesoscopic dimensions composed of protein-rich liquid exist in protein solutions, both in the homogeneous region of the solution phase diagram and in the region supersaturated with respect to an ordered solid phase, such as crystals; in the latter region they are crucial nucleation sites for ordered solids. We monitor, using three optical techniques, the long-term evolution of the clusters in lysozyme solutions at conditions where no condensed phases, liquid or solid, are stable or present as long-lived metastable domains. We show that cluster formation is a reversible process and that the clusters are in near equilibrium with the solution, up to a capillary correction. In contrast to classical phase transformations, the solution concentration at cluster-solution equilibrium is close to its initial value; this is akin to chemical reaction equilibria and demonstrates the complex chemical composition of the clusters. However, similar to classical phase transformations, en route to full equilibration, the average cluster size grows with time following a universal law t(0.26±0.03), independent of the cluster volume fraction; the cluster size distribution is scale-invariant at all stages of cluster evolution. Despite the correspondence of these behaviors to the Lifshitz-Slyozov-Wagner (LSW) theory predictions, the cluster sizes are about 10× smaller than the LSW prediction, likely due to the complex cluster composition. The observed cluster evolution helps us to understand nucleation mysteries, such as nucleation rates lower by orders of magnitude than classical theory predictions, nucleation rate variable under steady conditions, and others.

Protein-rich clusters of steady submicron size and narrow size distribution exist in protein solutions in apparent violation of the classical laws of phase equilibrium. Even though they contain a minor fraction of the total protein, evidence suggests that they may serve as essential precursors for the nucleation of ordered solids such as crystals, sickle-cell hemoglobin polymers, and amyloid fibrils. The cluster formation mechanism remains elusive. We use the highly basic protein lysozyme at nearly neutral and lower pH as a model and explore the response of the cluster population to the electrostatic forces, which govern numerous biophysical phenomena, including crystallization and fibrillization. We tune the strength of intermolecular electrostatic forces by varying the solution ionic strength I and pH and find that despite the weaker repulsion at higher I and pH, the cluster size remains constant. Cluster responses to the presence of urea and ethanol demonstrate that cluster formation is controlled by hydrophobic interactions between the peptide backbones, exposed to the solvent after partial protein unfolding that may lead to transient protein oligomers. These findings reveal that the mechanism of the mesoscopic clusters is fundamentally different from those underlying the two main classes of ordered protein solid phases, crystals and amyloid fibrils, and partial unfolding of the protein chain may play a significant role.

The two-step mechanism of nucleation of crystals in solutions posits that the formation of crystal nuclei occurs within structures of extended lifetimes, in which the nucleating solute is at high concentration. The validity of this mechanism has been demonstrated for proteins, small-molecule organic and inorganic materials, colloids, and polymers. Due to large molecule sizes, proteins are an ideal system to study the details of this nucleation pathway, in particular the formation mechanisms of the nucleation precursors and the associated physico-chemical rules. The precursors of protein crystal nuclei are protein-rich clusters of sizes ∼100 nm that contain 10,000-100,000 molecules and occupy less than 10(-3) of the total solution volume. Here we demonstrate, using oblique illumination microscopy, the liquid nature of the clusters of the protein lysozyme and reveal their inhomogeneous structure. We test a hypothesis put forth by theory that clusters primarily consist of transient protein oligomers. For this, we explore how varying the strength of the Coulomb interaction affects the cluster characteristics. We find that the cluster's size is insensitive to variations of pH and ionic strength. In contrast, the addition of urea, a chaotropic agent that leads to protein unfolding, strongly decreases the cluster size. Shear stress, a known protein denaturant, induced by bubbling of the solutions with an inert gas, elicits a similar response. These observations support partial protein unfolding, followed by dimerization, as the mechanism of cluster formation. The amide hydrogen-deuterium exchange, monitored by nuclear magnetic resonance, highlights that lysozyme conformational flexibility is a condition for the formation of the protein-rich clusters and facilitates the nucleation of protein crystals.