Surface and solvent influences on protein crystallization

The role of water in protein crystallization was explored by investigating the effects of three factors (salts, point mutations and pressure) on subtilisin crystallization.; Solubility and growth kinetics of three subtilisin mutants in three salt solutions were measured. The decrease of the solubility of Properase ® and Purafect® subtilisin followed the reverse order of the Hofmeister series: SCN− > NO3− > Cl−. The solubility of Properase® was higher than other two mutants. Crystal morphology changed with the nature of salts and the substitution of surface residues. The required supersaturation (c-s)/s for a given growth rate increased when solubility was decreased. The effect of anion on protein growth was related to the molar Gibbs free energy of hydration of the anion.; Structural and energetic considerations for crystallization of two subtilisin mutants (Properase® and Purafect®) were compared. The average hydrophobicity, solvent accessible surface area (ASA) and the number of hydrogen bonds and salt bridges were calculated to quantify surface properties of proteins in intermolecular contact patches. All three amino acid substitutions are present in the contact patches. Properase ® lattice involves more atomic contacts and hydrogen bonds and larger accessible surface area, which corresponding to the faster growth of Properase® crystals. Non-electrostatic interaction energy was calculated for each contact direction and the competition of misoriented molecules with correctly oriented ones was considered to explain the variation of growth kinetics; The increase of solubility with pressure gave a total volume change for crystallization of 37 cm3/mol, whereas the decrease of nucleation rate with pressure gave an activation volume for nucleation of 226 cm 3/mol. 983 water molecules were estimated to attend Properase ® crystallization.; The second virial coefficients (B₂) of Properase ® and Purafect® subtilisin under crystallization conditions were measured by static light scattering as a function of salt type and salt concentration, showing that conditions with slight negative B₂ are suitable for protein crystallization. A DLVO-type model was used to fit the effective Hamaker constants for subtilisin and solubility was quantitatively correlated with B₂ using a theoretically based correlation.