Chiara Piccolo

Experimental observations and numerical modelling of diffusion-driven crystallisation processes

This paper reports experimental results and modelling on the crystallisation processes induced by counter diffusion method of a precipitant agent in a lysozyme protein solution. Comparison between experimental observations and numerical simulations in the presence of convection and sedimentation and without them (suppressed using gel) provides a validation of the model. Different values of the initial protein concentration are used, in order to investigate the effects of supersaturation conditions on the process, and in particular on nucleation. The model and the experimental approach may represent a useful methodology for the determination of the parameters and conditions that may lead to protein crystallisation. A Mach-Zehnder interferometer is used to monitor the transport dynamics in situ in the fluid phase by observing the compositional field. The effect of the solute transport gives rise to a nucleation front that propagates inside the protein solution. The crystal formation, caused by progressing of the front, results in a modulation in time and in space (similar to Liesegang patterns), due to the non-linear interplay among transport, crystal nucleation and growth. Both experimental observation and numerical modelling show spatial and size distributions of crystals that demonstrate comparable evidences of the phenomena.

Effects of microgravity on the crystal quality of a collagen-like polypeptide

(Pro-Pro-Gly)(10) is one of the most widely studied collagen polypeptide models. Microgravity crystal growth of (Pro-Pro-Gly)(10) was carried out in the Advanced Protein Crystallization Facility aboard the Space Shuttle Discovery during the STS-95 mission. Crystals were successfully grown in all experiments, using both dialysis and free-interface diffusion methods. The quality of the microgravity-grown crystals and of ground-grown counterparts was assessed by X-ray synchrotron diffraction. Microgravity-grown crystals exhibited a significant improvement in terms of dimensions and resolution limit. As previously reported, crystals were orthorhombic, space group P2(1)2(1)2(1). However, the diffraction pattern showed weak reflections, never previously measured, that were consistent with new unit-cell parameters a = 26.9, b = 26.4, c = 182.5 A. This allowed the derivation of a new model for the arrangement of the triple-helical molecules in the crystals.

Higher modes of the mixed buoyant-Marangoni unstable convection originated from a droplet dissolving in a liquid/liquid system with miscibility gap

Some phenomena, never observed before, concerning a system composed by two organic-liquid bicomponent phases with a miscibility gap, used as transparent surrogates for immiscible metal alloys, are discussed and elucidated in the framework of experimental analyses and numerical simulations. It is shown that a single dissolving droplet at the bottom of a test cell behaves as an intriguing pattern-forming dynamical system leading to a wealth of different spatiotemporal modes of convection when the imposed temperature gradient is increased. The last part of the analysis is devoted to comparison with other similar phenomena (the flow instability pertaining to the Marangoni convection around bubbles surrounded by a liquid heated from above, and the case of rising buoyant jets), showing analogies and differences. Such a comparison is also used as a means to focus on the intrinsic nature of the present instability.

Crystallization of the collagen-like polypeptide (PPG)10 aboard the International Space Station. 1. Video observation.

Single chains of the collagen model polypeptide with sequence (Pro-Pro-Gly)(10), hereafter referred to as (PPG)(10), aggregate to form rod-shaped triple helices. Crystals of (PPG)(10) were grown in the Advanced Protein Crystallization Facility (APCF) both onboard the International Space Station (ISS) and on Earth. The experiments allow the direct comparison of four different crystallization environments for the first time: solution in microgravity ((g), agarose gel in (g, solution on earth, and gel on earth. Both on board and on ground, the crystal growth was monitored by a CCD video camera. The image analysis provided information on the spatial distribution of the crystals, their movement and their growth rate. The analysis of the distribution of crystals reveals that the crystallization process occurs as it does in batch conditions. Slow motions have been observed onboard the ISS. Different to Space-Shuttle experiment, the crystals onboard the ISS moved coherently and followed parallel trajectories. Growth rate and induction time are very similar both in gel and in solution, suggesting that the crystal growth rate is controlled by the kinetics at the interface under the used experimental conditions. These results provide the first data in the crystallogenesis of (PPG)(10), which is a representative member of non-globular, rod-like proteins.

Numerical and experimental analysis of periodic patterns and sedimentation of lysozyme

This paper deals with experimental investigation, mathematical modelling and numerical simulation of the crystallization processes induced by counter diffusion method of a precipitant agent in a lysozyme protein solution. Novel mathematical strategies are introduced to simulate the experiments and in particular to take into account the kinetics of the growth process and the motion of the crystals due to the combined effect of gravitational force and viscous drag if the sedimenting process is allowed (protein chamber free of gel). Comparison between experimental observations and numerical simulations in the presence of convection and sedimentation and without them provides a validation of the model. The crystal formation in gel results modulated in space. If the gel matrix is not present, convective cells arise in the protein chamber due to local inversions in the density distribution associated to nucleation phenomena. As time passes, these vortex cells migrate towards the top of the protein chamber exhibiting a different wave number according to the distance from the gel interface. The sedimentating particles produce a wake due to depletion of protein from the surrounding liquid. The models and the experiments may represent a useful methodology for the determination of the parameters and conditions that may lead to protein crystallization.

Video observation of protein crystal growth in the advanced protein crystallization facility aboard the space shuttle mission STS-95

Abstract Crystals of polypeptide with sequence (Pro-Pro-Gly) 10 were grown in the Advanced Protein Crystallization Facility during the US Space Shuttle STS-95 Mission. Well diffracting crystals were obtained by dialysis; they provided the highest resolution X-ray data so far collected for a collagen-like triple helical structure (Berisio et al., Acta Crystallogr. D 56 (2000) 55). During the Mission the crystal growth was monitored by a CCD video camera. The image analysis provided information on spatial distribution of crystals, their movements and their growth. The spatial distribution was non-uniform inside the reactors. Limited motions were observed. The linear growth rate indicates that, in some cases, the growth did not cease in the microgravity environment.

Interferometer for fluid physics experiments in microgravity environment

Several optical instruments are devoted for diagnostic in physico-chemical experiments, performed in microgravity environment. The aim of this paper is to describe a Wollaston interferometer devoted to measure the refraction index gradient of a transparent liquid media, in order to measure the concentration or temperature gradients around a formed drop. The Wollaston interferometer has been chosen because its sensitivity to the wavefront error can be tuned choosing the shearing component. Moreover this interferometer type has been chosen for his compactness and low sensitivity to the vibrations. This interferometer has been developed for the INEX MAM 3-Welcome project (EADS Space, Bremen / ESA contract), in which two different experiments, one devoted to the execution of the Marangoni migration, the other to the Wetting and Coalescence Prevention, have been carried out during the fly of MAXUS 5 Sounding Rocket.

Crystallization of the collagen-like polypeptide (PPG)10 aboard the International Space Station. 3. Analysis of residual acceleration-induced motion.

(PPG)(10) crystallization experiments onboard the ISS using the Advanced Protein Crystallization Facility have shown parallel and coherent crystal motions. The residual acceleration profiles and the History of the ISS Increment 3 mission allow a quantitative interpretation of these motions. Two events determine the observed crystal motions: the undocking of the Space Shuttle and a change in the ISS attitude required for power generation. No correlation between these motions and the crystal quality is apparent.