Comparison of solubilities and molecular interactions of BPTI molecules giving different polymorphs

International audienc

Solubility and prenucleation of aprotinin (BPTI) molecules in sodium chloride solutions

International audienc

Prenucleation, crystal growth and polymorphism of some proteins

International audienc

Crystal Growth Mechanism in a Solution of Hollow Whiskers of Molecular Compounds

International audienc

Crystal Growth Mechanism in a Solution of Hollow Whiskers of Molecular Compounds

International audienc

Comparison of solubility and interactions of aprotinin (BPTI) solutions in H2_2O and D2_2O

International audienceSmall-angle neutron scattering experiments are often performed with proteins solubilized in heavy water because of the large difference in neutron scattering properties of protons and deuterons. In order to characterize the effect of D$_2$O on physico-chemical properties of protein solutions, we investigated the effect of D$_2$O on the phase diagram and the interactions of bovine pancreatic trypsin inhibitor (BPTI) in solution. We measured the solubility in D$_2$O of BPTI solutions in the presence of NaCl (reverse solubility) and KSCN (direct solubility) and compared with the values measured by Lafont et al. in H$_2$O under the same conditions [Lafont et al., J. Crystal Growth 173 (1997) 132]. In the two salts, we found that BPTI solubility in D$_2$O is significantly lower than in H$_2$O. The curves representing the solubility of BPTI in KSCN are shifted by 7.2°C between light and heavy water, a shift obtained previously with lysozyme and representing the difference in the temperature of maximum density of both types of water [Gripon et al., J. Crystal Growth 177 (1997) 238; 178 (1997) 575]. In the case of BPTI in NaCl, we did not find this relationship between the solubility in H$_2$O and D$_2$O. We found, by dynamic light scattering, that BPTI attractive intermolecular interactions in the presence of NaCl in D$_2$O are significantly stronger than in H$_2$O. We investigated the association of BPTI molecules in crystallization conditions in the presence of NaCl in H$_2$O and D$_2$O by small-angle X-ray and neutrons scattering, respectively. In the presence of heavy water, the transition monomer–multimer is observed at about 2 mg/ml of BPTI in 1 M NaCl whereas in light water and in 1.4 M NaCl solution this transition is observed at about 15 mg/ml. These results clearly showed that BPTI in crystallization conditions is a multimer and confirm the importance of the isotopic nature of water in the crystallization of proteins. The replacement of H$_2$O by D$_2$O decreases the solubility and increases the attractive intermolecular interactions

The decameric structure of bovine pancreatic trypsin inhibitor (BPTI) crystallized from thiocyanate at 2.7 Å resolution

International audienceThe structure of a monoclinic form of bovine pancreatic trypsin inhibitor (BPTI) crystallized from a thiocyanate solution has been determined and refined at 2.7 Å resolution. The space group is P2$_1$ with $a$ = 71.56, $b$ = 73.83, $c$ = 64.47 Å, $\beta$ = 93.9° and Z = 20. The ten independent molecules were located by a multi-body molecular-replacement search as developed in the AMoRe program, starting from a single monomer model (PDB code: 6PTI). The molecular arrangement of the subunits is a decamer resulting from the combination of two orthogonal fivefold and twofold non-crystallographic axes. This builds a globular micelle-like particle which minimizes hydrophobic interactions with the solvent. The refinement was conducted with non-crystallographic symmetry constraints up to a final residual of R = 0.20 (R$_{free}$ = 0.26). The root-mean-square deviations from ideal geometry were 0.015 Å and 1.6° on bond distances and bond angles, respectively. Several sites for thiocyanate ions were analyze

Continuous and intensified membrane crystallization process : quality control of an active pharmaceutical ingredient

International audienc