Crystal phase formation in crowded lysozyme solutions

Abstract

Lysozyme crystallization in a crowded environment is investigated. Buffered protein solutions at pH 4.0 are used with no added precipitant (e.g. sodium chloride). Lysozyme concentrations over 250 mg ml⁻¹ and 0.31 M buffer salt (sodium acetate) are attained via equilibration of two microliter volume drops which possess different initial vapor pressure. The study demonstrates that gradual lysozyme crowding can lead to abrupt crystal phase formation by effective widening of the metastable zone and narrowing the nucleation zone towards the unstable region of the protein-salt phase diagram. The nucleation events occur perceivably in a cooperative manner. The crystal growth is rapid and finally the crystals occupy almost the whole drop volume. Virtually no temperature dependence of the crystal formation is observed in the interval 18–33 °C, except for the equilibration rate. The developed approach can be successfully used for adequate prediction of the time when crystal nucleation starts.