JuanMa Garcia-Ruiz

Agarose as crystallization media for proteins: I: Transport processes

A rheological and interferometric study of agarose sols and gels at different concentrations is reported. At concentrations smaller than 0.12% (w/v) agarose solutions behave like non-Newtonian fluids, while at higher concentrations they do so like viscoelastic gels. It is confirmed that agarose gels avoid mass and heat convective transport as well as sedimentation. We also find that agarose solutions at a concentration as low as 0.04% (w/v) are able to overcome buoyancy and crystal sedimentation. These results broaden the potential use of agarose in protein crystallization techniques.

Experimental evidence for the stability of the depletion zone around a growing protein crystal under microgravity

Experimental evidence is presented for the first time for the development and time evolution of concentration-depletion zones around protein crystals growing in microgravity and gelled on-ground experiments. Crystal motion and buoyancy-driven fluid movements as a result of residual accelerations and g-jitters are demonstrated to have an adverse effect on the stability of these depletion zones, provoking the breakdown of their radial symmetry. These findings may explain some of the controversial results reported on the quality of single crystals grown under microgravity in previous space missions.

Purification, crystallization and preliminary X-ray analysis of mexicain.

Mexicain is a 23.7 kDa papain-like cysteine protease from the tropical plant Jacaratia mexicana. Extracted as a mix of proteases from the latex of the fruit, mexicain is isolated after cation-exchange chromatography as the most abundant product. The purified product inhibited with E-64 was crystallized by sitting-drop vapour diffusion in the presence of ethanolamine. Cryoprotected crystals diffracted X-rays from a home source to 1.98 A and belong to the monoclinic space group P2(1), with unit-cell parameters a = 57.36, b = 90.45, c = 80.39 A, beta = 92.64 degrees . The asymmetric unit contains four molecules of mexicain, with a corresponding crystal volume per protein weight (V(M)) of 2.24 A(3) Da(-1) and a solvent content of 45% by volume. A molecular-replacement model has been determined and refinement is in progress.

Structure of the mexicain-E-64 complex and comparison with other cysteine proteases of the papain family.

Mexicain is a 23.8 kDa cysteine protease from the tropical plant Jacaratia mexicana. It is isolated as the most abundant product after cation-exchange chromatography of the mix of proteases extracted from the latex of the fruit. The purified enzyme inhibited with E-64 [N-(3-carboxyoxirane-2-carbonyl)-leucyl-amino(4-guanido)butane] was crystallized by sitting-drop vapour diffusion and the structure was solved by molecular replacement at 2.1 A resolution and refined to an R factor of 17.7% (R(free) = 23.8%). The enzyme belongs to the alpha+beta class of proteins and the structure shows the typical papain-like fold composed of two domains, the alpha-helix-rich (L) domain and the beta-barrel-like (R) domain, separated by a groove containing the active site formed by residues Cys25 and His159, one from each domain. The four monomers in the asymmetric unit show one E-64 molecule covalently bound to Cys25 in the active site and differences have been found in the placement of E-64 in each monomer.

Counterdiffusion protein crystallisation in microgravity and its observation with PromISS (Protein Microscope for the International Space Station)

The crystallisation by counterdiffusion is a very efficient technique for obtaining high-quality protein crystals. A prerequisite for the use of counterdiffusion techniques is that mass transport must be controlled by diffusion alone. Sedimentation and convection can be avoided by either working in gelled systems, working in systems of small dimensions, or in the absence of gravity. We present the results from experiments performed on the ISS using the Protein Microscope for the International Space Station (PromISS), using digital holography to visualise crystal growth processes. We extensively characterised three model proteins for these experiments (cablys3*lysozyme, triose phosphate isomerase, and parvalbumin) and used these to assess the ISS as an environment for crystallisation by counterdiffusion. The possibility to visualise growth and movement of crystals in different types of experiments (capillary counterdiffusion and batch-type) is important, as movement of crystals is clearly not negligible.

Protein crystallisation under microgravity conditions: What did we learn on TIM crystallisation from the Soyuz missions?

This study deals with heat transfer enhancement surface manufactured by thermal spraying. Two thermal spraying methods using copper as a coating material, wire flame spraying (WFS) and vacuum plasma spraying (VPS), were applied to the outside of copper cylinder with 20 mm OD. The surface structure by WFS was denser than that by VPS. The effect of gravity on boiling heat transfer coeffcient and wall superheat at the onset of boiling were experimentally evaluated under micro- and hyper-gravity condition during a parabolic trajectory flight of an airplane. Pool boiling experiments in saturated liquid of HCFC123 were carried out for heat fluxes between 1.0 and 160 kW/m2 and saturated temperature of 30 °C. As a result, the surface by VPS produced higher heat transfer coefficient and lower superheat at the onset of boiling under microgravity. For the smooth surface, the effect of gravity on boiling heat transfer coefficient was a little. For the coating, a large difference in heat transfer coefficient to gravity was observed in the moderate heat flux range. The heat transfer coefficinet decreased as gravity changed from the normal to hypergravity, and was improved as gravity changed from the hyperto microgravity. The difference in heat transfer coefficient between the normal and microgravity was a little. Heat transfer enhancement factor was kept over the experimental range of heat flux. It can be said that boiling behavior on thermal spray coating might be influenced by flow convection velocity.

Crystallization screening directly in electrophoresis gels

Screening of crystallization conditions can be directly performed within electrophoretic gels run during the purification steps. The technique is demonstrated for different methods of electrophoretic separations and for two different gels, agarose and polyacrylamide. Two different techniques are proposed to perform the screening: direct diffusive mixing in small gel slices and diffusion technique in gel bands.

Fluid patterns in the diffusive field around a growing crystal

Abstract The use of simultaneous diffusion of particles in a modified DLA system [Witten and Sander, Phys. Rev. Letters 47 (1981) 1400; Phys. Rev. B 27 (1983) 5686], leading to several morphologycal outputs, permitted us to observe the diffusive field around the growing cluster. We show that the existence of morphological instabilities produces the symmetry breaking of the diffusive field around the cluster and the formation of preferred flow induced by a morphological instability.

THE ANISOTROPY OF ON-LATTICE SIMULATIONS OF AGGREGATE GROWTH

The origin of anisotropy in on-lattice simulations of cluster growth is investigated by quantifying the anisotropic behavior of the two processes involved in the simulation: transport and aggregation of nutrient particles. Three different anisotropic effects are described: anisotropic surface kinetics, anisotropic concentration profiles and anisotropic screening. The relevance of these sources of anisotropy in natural systems is discussed.