Protein Conformational Flexibility Enables the Formation of Dense Liquid Clusters: Tests Using Solution Shear

According to recently proposed two-step nucleation mechanisms, crystal nuclei form within preexisting dense liquid clusters. Clusters with radii about 100 nm, which capture from 10(-7) to 10(-3) of the total protein, have been observed with numerous proteins and shown to host crystal nucleation. Theories aiming to understand the mesoscopic size and small protein fraction held in the clusters have proposed that in solutions of single-chain proteins, the clusters consist of partially misfolded protein molecules. To test this conjecture, we perturb the protein conformation by shearing solutions of the protein lysozyme. We demonstrate that shear rates greater than a threshold applied for longer than 1 h reduce the volume of the cluster population. The likely mechanism of the observed response involves enhanced partial unfolding of lysozyme molecules, which exposes hydrophobic surfaces between the constituent domains to the aqueous solution.