Understanding the crystallisation of an acidic protein by dilution in the ternary NaCl-2-methyl-2,4-pentanediol-H2O system

Halophilic malate dehydrogenase is a negatively charged protein that crystallises well in a dilution process following a complex interplay with the three components of NaCl-MPD-H2O solvents (MPD: 2-methyl-2,4-pentanediol). The process was characterised by measuring the folding state of the protein, its concentration, its apparent solubility and second virial coefficients in various NaCl-MPD-H2O ratios representative of the phase diagram of the system. The protein crystallises by vapour diffusion between a drop containing protein in aqueous NaCl and MPD and a bath containing a given percentage of MPD in water. The starting drop is bi-phasic, in which the protein is concentrated in the salt-rich phase. This phase evolves towards a solution less concentrated in both NaCl and protein but more concentrated in MPD, before the system is driven to a single-phase region, where crystals are obtained. The protein stability is preserved during its crystallisation. We showed from second virial coefficient measurements that crystal formation is correlated with a slow evolution from repulsive to attractive protein-protein interactions. The end of the vapour diffusion process between the drop and the bath corresponds to a decrease of the attractive interaction, which we suggest favours crystal growth. The application of these results to crystallisation of other charged proteins and nucleic acids is discussed. (C) 2001 Elsevier Science B.V. All rights reserved.