Identifying nucleation temperatures for lysozyme via differential scanning calorimetry

Production of diffraction-quality protein crystals mandates nucleation of a moderate number of crystals. Excessive nucleation can lead to many small crystals of poor morphology, making them useless for structural determination via X-ray crystallography. In this work, differential scanning calorimetry (DSC) and light scattering are utilized to delineate the liquid–liquid binodal in lysozyme solutions at concentrations typically used for crystal growth (10–50 mg/ml). Such cloud point temperatures are determined over a range of protein concentrations and compared to visually observed nucleation temperatures. In all cases, nucleation occurs at temperatures above the cloud point temperature. Thus, measurement of the cloud point temperature gives the crystal grower a bound on the protein crystal growth problem. Nucleation is reproducibly induced within 2°C of the cloud point temperature; while prior work indicates that growth, when carried out near the saturation temperature, yields high quality crystals. The ease of measuring the cloud point via turbidity measurements allows for the rational selection of an initial temperature to induce protein nucleation.