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.

Slow-Down in Diffusion in Crowded Protein Solutions Correlates with Transient Cluster Formation

“…Other studies show that the lifetime of lysozyme clusters is finite on millisecond time scales 77, 78 . The presented lifetimes are also not in contradiction with reports for solutions of bovine β-lactoglobulin (BLG) that indicate that BLG clusters are static on NSE observation time scale, i.e.…”

Phase equilibrium and mechanisms of crystallization in liquid–liquid phase separating system

“…Liquid-liquid equilibrium data have been investigated for some "anti-solvent" crystallization experiments [2,3], spherical crystallizations using a three solvents system [4,5], oiling out phenomena [6][7][8][9][10] and reverse addition processes [11]. The LLPS makes crystallization difficult in protein systems [12,13], prevents drug nucleation [14][15][16], and hinders nucleation in crystallization of droplets [17]. Lack of phase diagram data for crystallization processes with LLPS makes it difficult to understand, design and control the process.…”

Macromolecular Crystallization Controlled by Colloidal Interactions: The Case of Urate Oxidase

“…Lysozyme has long been the model protein most often chosen to study nucleation and crystal growth mechanisms (Drenth, 2005;Gavira & Garcia-Ruiz, 2009;Liu et al, 2010;Vekilov, 2010), since it is easily available at low cost. We used urate oxidase from Aspergillus flavus as a new model system to explore the crystallogenesis of proteins in general and the crystallographic structure of urate oxidase in particular.…”