Jean-Pierre Astier

Crystallization mechanisms in solution

Abstract Crystallization from solution is a sequence of events which occur more or less consecutively but are rarely completely unconnected. The present contribution is a survey of nucleation, growth, phase transition, habit modification and ripening. Its aim is to provide a theoretical basis to biochemists who intend to approach crystallization.

α-amylase crystal growth investigated by in situ atomic force microscopy

Abstract The growth behavior of porcine pancreatic α-amylase at defined supersaturation has been investigated by means of temperature controlled in situ atomic force microscopy (AFM). The step velocities measured by AFM were in overall agreement with the normal growth rates of an individual face measured by optical microscopy. In addition, highly local growth dynamics could be visualized. Imaging in tapping mode revealed crystalline amylase aggregates attached to the basal face and their subsequent incorporation into growing terraces producing a macrodefect. At high supersaturation ( β =1.6) 2-D nucleation was found to be the dominating growth mechanism, whereas at lower supersaturation ( β =1.3) the growth process appears to be defect controlled (spiral growth). The analysis of step heights on 2-D nucleation islands (monomolecular protein layers) and growth steps (two molecules in height) in combination with results from light scattering experiments suggest that a single protein molecule is the basic growth unit.

Calcium Carbonate Crystals Promote Calcium Oxalate Crystallization by Heterogeneous or Epitaxial Nucleation: Possible Involvement in the Control of Urinary Lithogenesis

A large proportion of urinary stones have calcium oxalate (CaOx) as the major mineral phase. In these stones, CaOx is generally associated with minor amounts of other calcium salts. Several reports showing the presence of calcium carbonate (CaCO3) and calcium phosphate in renal stones suggested that crystals of those salts might be present in the early steps of stone formation. Such crystals might therefore promote CaOx crystallization from supersaturated urine by providing an appropriate substrate for heterogeneous nucleation. That possibility was investigated by seeding a metastable solution of45Ca oxalate with vaterite or calcite crystallites. Accretion of CaOx was monitored by45Ca incorporation. We showed that (1) seeds of vaterite (the hexagonal polymorph of CaCO3) and calcite (the rhomboedric form) could initiate calcium oxalate crystal growth; (2) in the presence of lithostathine, an inhibitor of CaCO3 crystal growth, such accretion was not observed. In addition, scanning electron microscopy demonstrated that growth occurred by epitaxy onto calcite seeds whereas no special orientation was observed onto vaterite. It was concluded that calcium carbonate crystals promote crystallization of calcium oxalate and that inhibitors controlling calcium carbonate crystal formation in Henle’s loop might play an important role in the prevention of calcium oxalate stone formation.A large proportion of urinary stones have calcium oxalate (CaOx) as the major mineral phase. In these stones, CaOx is generally associated with minor amounts of other calcium salts. Several reports showing the presence of calcium carbonate (CaCO3) and calcium phosphate in renal stones suggested that crystals of those salts might be present in the early steps of stone formation. Such crystals might therefore promote CaOx crystallization from supersaturated urine by providing an appropriate substrate for heterogeneous nucleation. That possibility was investigated by seeding a metastable solution of45Ca oxalate with vaterite or calcite crystallites. Accretion of CaOx was monitored by45Ca incorporation. We showed that (1) seeds of vaterite (the hexagonal polymorph of CaCO3) and calcite (the rhomboedric form) could initiate calcium oxalate crystal growth; (2) in the presence of lithostathine, an inhibitor of CaCO3 crystal growth, such accretion was not observed. In addition, scanning electron microscopy demonstrated that growth occurred by epitaxy onto calcite seeds whereas no special orientation was observed onto vaterite. It was concluded that calcium carbonate crystals promote crystallization of calcium oxalate and that inhibitors controlling calcium carbonate crystal formation in Henle’s loop might play an important role in the prevention of calcium oxalate stone formation.

BPTI liquid-liquid phase separation monitored by light and small angle X-ray scattering.

In the field of protein crystallization, a better knowledge of the nucleation process is essential to control the nucleation rate, the growth and therefore the size and the quality of crystals. With that aim, it becomes clear that the important stage is the determination of the protein phase diagram. We highlighted and investigated the bovine pancreatic trypsin inhibitor (BPTI) binary liquid-liquid phase separation in 350 mM KSCN solutions as a function of temperature. We measured the low concentration part of the binodal curve using light scattering and optical microscopy. We show, from small angle X-ray scattering experiments, that the high concentrated phase sediments in the bottom of the capillary and we analysed the low concentrated phase in terms of monomers/decamers equilibrium.

Influence of polydispersity on protein crystallization: a quasi-elastic light-scattering study applied to α-amylase

The early stages of the crystallization process of porcine pancreatic alpha-amylase were investigated by quasi-elastic light scattering. It is shown that at 288 and 293 K the diffusion coefficient does not monotonically change with increasing protein concentration but passes through a maximum at 10 mg ml(-1). In supersaturated solutions, prior to nucleation, the protein is strictly monodisperse. Nucleation induces the formation of aggregates and a polydispersity of, for example, 18% for an initial supersaturation C/C(e) = 5.8. Monodispersity is restored after the nuclei have grown and partially consumed the solute. On the other hand, polydispersity increases up to 20% at 298 K if the protein concentration decreases to 3-4 mg ml(-1), values at which the solutions are under-saturated. When the protein concentration exceeds 5-6 mg ml(-1) the protein becomes monodisperse again. These results, confirmed by those of another system we are studying (bovine pancreatic trypsin inhibitor), are at variance with the statements that supersaturation is always at the origin of aggregation and polydispersity, and that in undersaturated solutions the diffusion coefficient should remain constant for obtaining crystals once the solutions are supersaturated.

pH-dependent oligomerization of BPTI in undersaturated and supersaturated solutions studied by dynamic light scattering

Bovine pancreatic trypsin inhibitor (BPTI), a small protein molecule, is reported to form several types of oligomers in solution including a decamer. We investigate in this work the relation between the oligomerization and crystallization. The oligomerization and interaction between BPTI molecules in NaCl and NaH2PO4/K2HPO4 solutions were investigated by dynamic light scattering (DLS). In the NaCl solutions, we found that strong electrostatic interaction amongBPTI and chloride ions led to the decamer formation only at an acidic pH, whereas the protein solutions were monomeric at a neutral or basic pH, where the electrostatic interaction seemed to be unimportant. The single crystals with a hexagonal truncated bipyramidal shape grew only in the decameric solutions at an acidic pH in the presence of NaCl. On the other hand, the electrostatic interaction was weak in the presence of NaH2PO4/K2HPO4 at all the pH values investigated, and the solutions were always monomeric. In contrast with the NaCl solutions, single crystals with a rod-like shape appeared at a neutral and basic pH in the NaH2PO4/K2HPO4 solutions. These results suggest that the crystallization mechanism in the presence of NaCl, where electrostatic interactions seem to drive crystallization, is totally different from that in the presence of NaH2PO4/K2HPO4 where another interaction such as hydrophobic interaction or hydrogen bonding might cause crystallization. r 2002 Elsevier Science B.V. All rights reserved.

Characterization and crystallization of the Endoglucanase A from Clostridium Cellulolyticum in solution

This study deals with the Endoglucanase of the family A (CelCCA) from Clostridium Cellulolyticum (MW = 43 kDa. pI≅6), CelCCA takes part in a multienzyme complex (cellulosome) including a majority of cellulases whose detailed molecular organization and mode of action are not well known. The aim of this work is to understand the crystal growth mechanisms of CelCCA, Solubilities and crystal growth rates were measured, and the behavior of CelCCA molecules in under and supersaturated solutions was investigated. The presence of different isoenzymes in the CelCCA solution was observed by mass spectroscopy and the presence of aggregates was detected by dynamic light scattering. It was shown that these aggregates, due to the different isoenzymes of CelCCA, hinder the crystallization of CelCCA. Interactions between protein molecules, measured by small-angle X-ray scattering, were found attractive under crystallization conditions (14% PEG 4000, 90 mM sodium citrate, pH 6). The addition of CaCl 2 to the solution was found to improve the crystallization, favoring the salting-out effect, but had no influence on the solubility and on the structure of CelCCA. A new polymorph of CelCCA was found, and the solubility of the two polymorphs was measured. The two polymorphs, which grew under similar conditions, differ mainly by their crystallographic b parameter, the space group P2 1 2 1 2 being conserved. Growth rates were measured, it was found that the more soluble polymorph has a higher growth rate.

Crystallization of a recombinant form of the complete sequence of human γ‐interferon: characterization by small‐angle X‐ray scattering, mass spectrometry and preliminary X‐ray diffraction studies

The crystallization conditions of a recombinant form of the complete sequence of human gamma-interferon, designated r-hu IFN-gamma (RU 42369), have been determined after studying the behaviour of this protein in solution by small-angle X-ray scattering (SAXS) as a function of pH and salt type. IFN-gamma is difficult to crystallize without truncating at least the last five amino acids of the C-terminus; the SAXS results suggest viable crystallization conditions that led to crystals of r-hu IFN-gamma suitable for X-ray diffraction analysis. The crystals were grown in the presence of ammonium sulfate using vapour-diffusion techniques. The crystals, which diffract to 5 A resolution at best, belong to the primitive tetragonal space group P42(1)2 and have unit-cell parameters a = b = 123.4, c = 93.4 A. The protein contained in these crystals was analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), which verified the presence of the complete amino-acid sequence of r-hu IFN-gamma.