Luigi Carotenuto

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.

Lysozyme crystal growth kinetics in microgravity.

Mach-Zehnder interferometry is applied to quantitatively characterize growth of lysozyme crystals in microgravity. Experiments were performed by the Free Interface Diffusion technique into APCF FID reactors using large seeds. Tracking of the experiments using interferometry allowed to monitor the onset of supersaturation and the seed growth. A large and stable concentration depletion zone around the growing crystal developed, whose time evolution was analyzed. The interferograms were analyzed taking into account finite thickness of the cell by integrating the concentration over the straight lines through the optical path. It was concluded that there may be a quasi-steady state growth mode at the stage when the spacial concentration distribution did not change but its absolute value over all the cell was slowly diminishing. From this portion of the data, an estimate was made of the dimensionless parameter beta R/D where beta is the face kinetic coefficient, R is the effective crystal size and D is the lysozyme diffusivity in solution, as followed from the steady state model. For the assumed quasi steady state data portion, the parameter varies between 0.7 and 0.9 suggesting mixed diffusion-interface kinetic controlled growth.

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.

Effect of convective disturbances induced by g-jitter on the periodic precipitation of lysozyme

Numerical simulations are carried out to investigate the crystallization process of a protein macromolecular substance under two different conditions: pure diffusive regime and microgravity conditions present on space laboratories. The configuration under investigation consists of a protein reactor and a salt chamber separated by an “interface”. The interface is strictly related to the presence of agarose gel in one of the two chambers. Sedimentation and convection under normal gravity conditions are prevented by the use of gel in the protein chamber (pure diffusive regime). Under microgravity conditions periodic time-dependent accelerations (g-jitter) are taken into account. Novel mathematical models are introduced to simulate the complex phenomena related to protein nucleation and further precipitation (or resolution) according to the concentration distribution and in particular to simulate the motion of the crystals due to g-jitter in the microgravity environment. The numerical results show that gellified lysozyme (crystals “locked” on the matrix of agarose gel) precipitates to produce “spaced deposits”. The crystal formation results modulated in time and in space (Liesegang patterns), due to the non-linear interplay among transport, crystal nucleation and growth. The propagation of the nucleation front is characterized by a wavelike behaviour. In microgravity conditions (without gel), g-jitter effects act modifying the phenomena with respect to the on ground gellified configuration. The role played by the direction of the applied sinusoidal acceleration with respect to the imposed concentration gradient (parallel or perpendicular) is investigated. It has a strong influence on the dynamic behaviour of the depletion zones and on the spatial distribution of the crystals. Accordingly the possibility to obtain better crystals for diffraction analyses is discussed.

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.

Numerical simulation of FID hydrodynamics of protein crystallization

Abstract Recently, some of us have proposed predictive equations for the four diffusion coefficients, Dij, in a ternary system of hard spheres. These equations have been tested successfully for some ternary systems of interest in protein crystallization: mixture of PEGs in aqueous solution, and the system lysozyme–NaCl–H2O. These equations supply the tools for a correct coupled analysis of the diffusion phenomena occurring in crystallization processes. Using the previous approach for the dependence of the four diffusion coefficients on the solute concentrations, a numerical analysis of transport phenomena in a model system that can simulate protein crystal growth has been carried out. The set of equations is based on the incompressible form of the two-dimensional and time dependent Navier–Stokes equations. The hypothesis of variable thermodynamic properties (diffusion and dynamical viscosity) and the Bousinnesq approximation for the momentum equation have been considered. The study has been performed by means of a numerical code based on a finite difference method. The influence of the coupled diffusion has been evaluated at different gravity levels.

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.

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.

Numerical simulation of three-dimensional thermocapillary flows in liquid bridges

Instability of thermocapillary convection has been studied by using a numerical code based on a control volume method. The momentum, continuity, and energy equations are solved in a cylindrical configuration with the thermocapillary boundary conditions at the free surface. The convective fluxes are calculated via an upwinding method, and the numerical values are updated via a single-step time marching method. We report the results obtained for two different Prandtl numbers (1 and 32). The disturbance growth has been determined during the transition from the axisymmetric to the oscillatory three-dimensional flow; the oscillatory modes, critical Reynolds numbers, and frequencies are compared with those obtained by stability analyses. Two different regimes occur in supercritical conditions: the oscillations initially emerge as a pulsating regime and, successively, become an azimuthally rotating structure. The latter is also subjected to further transitions characterized by an increase of the number of fundam...