(592e) Mesoscopic Clusters in Protein Solutions: Intermolecular Forces and Chemical Mechanisms

According to the recently proposed two-step nucleation mechanism, protein-rich liquid clusters are essential precursors of ordered solid protein phases.  Such clusters with mesoscopic sizes of about 100 nm and macroscopic lifetimes have been demonstrated in solutions of about ten diverse proteins under conditions far removed from liquid-liquid co-existence.  The understanding of the mechanism of cluster formation had proven a hard challenge.  Towards the sought insights, we monitor cluster-containing protein solutions using dynamic and static light scattering and determine cluster‘s sizes, the fraction of the solution volume that they occupy, and their concentration.  We correlate these cluster characteristics to the osmotic second virial coefficient B₂, a measure of intermolecular interactions in the studied solutions.

Hydrophobic forces contribute significantly to protein stability and aggregation.  To test their role in cluster formation, we monitor clustering behavior in lysozyme solutions in the presence of urea at concentrations up to 2 M; this upper limit is well below the concentration required to fully denature proteins, 7 – 8 M.  The results on the cluster behavior indicate that urea mostly acts to weaken the attraction between hydrophobic patches either on the molecular surface or those exposed to the solution after partial protein unfolding. 

For further tests of the hydrophobic interactions, we gentle bubble N₂, He, and air through the solution.  Due to the shear forces caused by bubbling, proteins may partially unfold and expose their hydrophobic residues to the solvent.  Besides exploring the role of hydrophobic forces, bubbling tests the significance of chemical mechanisms contributing to cluster formation.  Bubbling with inert gasses such as He or N₂ lowers the amount of the dissolved O₂ and the solution oxidative potential; while air bubbling increases it.  Oxidative stress may contribute to aggregation by activating the sulfide groups and enabling intermolecular disulfide bridges.  These experiments reveal that partial protein unfolding is a part of the cluster mechanism. 

A recent theoretical model posits that the presence of protein complexes (in a simple case, dimers) is a requirement for cluster formation.  The bubbling results strongly suggest that the dimers form due to the saturation of the hydrophobic surfaces exposed to the solution after unfolding.  Additional support for this notion comes from experiments with 2-mercaptoethanol, a reducing agent which disrupts the intramolecular ~S-S~ bonds and breaks the tertiary structure of protein molecules.  The addition of 2-mercaptoethanol induces cluster behaviors similar to those of bubbling.

The experiments above strongly support the idea that partial protein unfolding is the main mechanism of cluster formation.  To specify the locations along the protein main chain involved in unfolding, we characterized the degree of unfolding of the aminoacid residues from the rate of exchange of H to D measured in nuclear magnetic resonance experiments.  We map the residues experiencing faster hydrogen-deuterium exchange in clusters containing solution and compare them to those in a cluster free sample.  The results indicate that the residues that are more unfolded in the clusters are located predominantly in hinges regions of the protein chain.