Toward Further Understanding of Lysozyme Crystallization: Phase Diagram, Protein−Protein Interaction, Nucleation Kinetics, and Growth Kinetics

Abstract

A detailed study on the phase behavior and mechanism of lysozyme crystallization is presented. The nucleation and crystal growth rates, crystal morphologies, solubility, second virial coefficients, and cloud-point temperatures under different solution conditions were experimentally measured and theoretically analyzed. The nucleation rates were found to increase with the protein concentration and sodium chloride concentration when crystallization occurred in the solid−liquid coexistence region, and the dependence of nucleation rate on protein concentration was identified to bifurcate into two groups around the liquid−liquid coexistence boundary. The suppression of the nucleation in the protein-rich phase was investigated in terms of protein−protein interactions determined by Raman microscopy and the self-assembly of the protein molecules. The concentration distribution in the region in between the growing nucleus and the protein-rich phase was derived to explain the increase of nucleation rate in the liquid−liquid coexistence region. The different shapes of lysozyme crystals were obtained in different lysozyme and NaCl concentration regions. The growth rates of the (110) and (101) faces of tetragonal lysozyme crystals were also determined. These new observations and analysis were expected to provide further understanding and guidelines for protein crystallization.