(731e) Systematic Investigation of the Nucleation Mechanism of Lysozyme Crystallization In the Liquid-Liquid Co-Existence Region

Previous work within this group has highlighted that both liquid-liquid phase separation and crystallization occur in protein solutions. The liquid-liquid phase separation and the crystal nucleation and growth processes observed and recorded by an optical microscope suggested that two different nucleation pathways may exist. The current research aims to assess the nucleation of lysozyme crystals in the liquid-liquid co-existence region systematically. The nucleation kinetics has been investigated by the method of initial rates. Micro-batch crystallization experiments were conducted at constant temperature, 10 degree Celsius, with different supersaturations. An optical microscope with a heating/cooling stage was applied to determine the liquid-liquid co-existence curve, measure the initial nucleation rate and observe the liquid-liquid phase separation and subsequent crystallization process. It was found that the nucleation was arrested in the protein-rich phase. The crystals nucleated from the protein-lean phase. As the crystal nucleated from the solution, the concentration of the protein-lean phase dropped, the protein rich phase dissolved to replenish the supersaturation. By considering the buffering zone effect caused by liquid-liquid phase separation, the nucleation kinetics data were modeled using classical nucleation theory. The nucleation rate was found to increase with the protein concentration as expected.

In addition, the conformation changes and the related protein-protein interactions of lysozyme in aqueous solutions, protein-rich phase, protein-lean phase and crystalline state were studied by Raman microscope. The Raman spectra of lysozyme molecules in different states proved the proposed nucleation mechanism from the view of protein conformation. There was a good agreement among the Raman spectra of lysozyme molecules in different states in amide I and amide III regions, which suggested that lysozyme molecules retained their backbone structure upon aggregation or crystallization. However, the changes in the tyrosine doublet positions and relative intensity revealed that the side chain conformation changed under different protein-protein interactions. The doublet ratio increases when approaching the condition of crystallization with the highest doublet ration observed in crystal. The high doublet ratio was attributed to the exposure of the originally buried tyrosine residue to the outer surface of protein molecule to involve in intermolecular hydrogen bonding, which may increase the protein-protein interaction. The doublet ratio of the protein-lean phase was similar to that of the protein crystallization solution without liquid-liquid phase separation. The doublet ratio of the protein rich phase was larger than that of the protein crystallization solution, which suggested a stronger protein-protein interaction. In protein-lean phase, the relative weaker interactions enabled the protein molecules to adapt their orientations as they did in the crystal more easily. On the contrary, in protein-rich phase, the stronger interactions may take a certain period of time for the protein molecules to change their conformation and orientation, which may be the reason of the arrestment of the nucleation in the protein-rich phase.

In summary, this detailed experimental and theoretical study provides further understanding of the mechanism of protein nucleation in the liquid-liquid co-existence region.