Ostwald-Like Ripening of the Anomalous Mesoscopic Clusters in Protein Solutions

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.

Role of clusters in nonclassical nucleation and growth of protein crystals

“…Secondly, it is striking that we find such a strong dependence of the nucleation kinetics--and the impact of filtration thereon--on protein stock solution age. This is reminiscent of two recent papers that have revealed that protein clusters undergo a gradual maturation, i.e., the mean cluster size grows over time (21,41). However, they report on processes operating on a timescale of hours and days, certainly not weeks and months, as is the case here.…”

“…The dependence of protein stock solution age on the crystallization outcome is perhaps not entirely unexpected. Works in the past have reported cluster maturation (i.e., increase in size) through an Ostwald-like ripening process (21,41). We also observe that the reduction in nucleation rate becomes more pronounced at higher σ-values--the nucleation rate drops to a basal level after filtration which is independent of σ. σ was varied by increasing the bulk protein concentration, and thus by extension, the cluster number density in the mother liquor.…”

Mechanism for the stabilization of protein clusters above the solubility curve

“…the growth of larger clusters at the expense of smaller ones. Something like ripening has in fact been reported by Li et al [9] albeit with the unusual feature that the ripening stops while there is still a finite population of clusters. If the present model were correct, this "ripening" would have to be reinterpreted: perhaps as a slowly relaxing transient.…”

Mesoscopic Liquid Clusters Represent a Distinct Condensate of Mutant p53

“…2,34 The combination of weaker binding and structural destabilization classifies R248Q among the strongest predictors of patient death in ovarian cancer. 35 We examine the existence of two distinct liquid phases: macroscopic liquid, similar to the phase found with other partially disordered proteins, [17][18][19][20][21] and mesoscopic protein-rich clusters observed with numerous globular proteins [23][24][25]36,37 and recently found for wild type p53. 38 We establish that p53 R248Q forms mesoscopic clusters, which are distinct form the macroscopic dense liquid, and that they host the nucleation of amyloid fibrils.…”