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

Abstract

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.

Weakly-bound Dimers that Underlie the Crystal Nucleation Precursors in Lysozyme Solutions

“…Exploring the puzzling cluster behaviors revealed that whereas the volume occupied by the clusters population is dictated by the thermodynamics of the protein solution, the size of the clusters reflects the dynamics of formation and decay of protein complexes (32,37,39,40). The complexes must be weakly-bound and decay after a certain lifetime (32,33,40,42).…”

“…for single-chain protein, or misassembled oligomers of multiple-chain proteins (23,43). Characterization of the cluster populations in lysozyme solutions after destabilization of the protein conformations with urea (40,44) and in sheared solutions (39,45) suggest that enhanced partial unfolding of lysozyme molecules is a part of the mechanism of lysozyme clusters. Such unfolding exposes hydrophobic surfaces between the lysozyme constituent domains to the aqueous solution and facilitates the formation of domain-swapped dimers, Scheme 1a.…”

“…Previous analyses of commercial lysozyme preparations have suggested the presence of up to five additional proteins with molecular weights 18,28,39,66,[78][79][80][81][82]. Whereas the 28 kDa heterogeneity was tentatively identified as a lysozyme dimer produced by oxidation, the identity of the other polypeptides was not firmly established (80).…”

“…Their diameters are of order 100 nm (22,26,34,35) and each cluster contains 10 4 -10 5 protein molecules (26, 32-34, 36, 37). With lysozyme, a robust protein used as a model in several biophysics fields, cluster formation is reversible and the clusters exchange protein with the host solution (33,(37)(38)(39)(40). Remarkably, the responses of the characteristic cluster size and phase volume to variations of the ionic strength, pH, and additive concentration are decoupled and the cluster size is independent of protein concentration, solution ionic strength, and pH (39,40).…”

“…With lysozyme, a robust protein used as a model in several biophysics fields, cluster formation is reversible and the clusters exchange protein with the host solution (33,(37)(38)(39)(40). Remarkably, the responses of the characteristic cluster size and phase volume to variations of the ionic strength, pH, and additive concentration are decoupled and the cluster size is independent of protein concentration, solution ionic strength, and pH (39,40). These findings are in sharp contrast to observations for typical phase transitions, in which the size of the incipient domains and the volume occupied by the emerging phase increase or decrease concurrently (41).…”