Effect of high hydrostatic pressure on nucleation and growth of protein crystals

Abstract

The influence of hydrostatic pressure on the nucleation and growth of protein crystals was studied. A micromethod was developed to establish a solubility phase diagram of hen egg-white lysozyme as a function of pressure and protein concentration. The pressure dependence of the formation of canonical tetragonal crystals was investigated at different precipitating agent and protein concentrations (in the range 0.6–1.2M NaCl and 10–35 mg/ml lysozyme). The apparent protein solubility significantly increases when pressure is raised from 0.1 MPa (atmospheric pressure) to 250 MPa. With an increase in pressure, the size and number of lysozyme crystals decline and a transition to urchin-like particles made of crystalline needles progressively occurs. The shape of tetragonal crystals becomes more elongated in a limited region of the phase diagram as indicated by the ratio of the lengths of the (110) and (101) faces. Single tetragonal crystals grown under high pressure diffract X-rays at high resolution. They belong to the same space group and have identical cell parameters as control crystals grown at atmospheric pressure. Changes in solubility and crystallizability are explained by pressure-induced minor reversible alterations in the protein structure.