Juan Ma. García-Ruiz

A supersaturation wave of protein crystallization

A microgravity protein crystallization experiment is described in which the existence of a supersaturation wave traveling across a diffusion-reaction system is experimentally demonstrated for the first time. The self-organized dynamics of the experimental setup were used to implement a crystallization technique able to search automatically through the crystallization parameter space for optimum nucleation and growth conditions. The crystals obtained by this automatic optimization produced the highest quality X-ray diffraction data ever collected from the model protein used in the experiment.

Granada Crystallisation Box: a new device for protein crystallisation by counter-diffusion techniques.

Granada Crystallisation Box (GCB) is a new crystallisation device designed to perform counter-diffusion experiments. Here we describe the device and its use for protein crystallisation purposes. GCB allows one to explore and exploit the coupling between crystallisation and diffusion. The role of viscous fluids, gels and/or microgravity can be enhanced by using capillary volumes, creating a perfect diffusive mass transport scenario. The use of capillaries also reduces the consumption of macromolecules and avoids the handling of crystals for X-ray diffraction data collection.

Reinforced protein crystals

Large tetragonal hen egg white lysozyme single crystals (up to 16 mm 3 ) can be obtained by the counter-diffusion method, using high concentration silica gels. The protein crystal lattice is able to incorporate large amounts of silica while still maintaining its short-range crystallographic order. The crystal morphology is controlled by the concentration of the silica gel, which can reduce surface energy anisotropy to such an extent that spherical single crystals can be obtained as growth forms. The mechanical properties and the stability of the crystals against dehydration are improved by the incorporated hydrophilic silica polymeric network. This makes it possible to record full diffraction data sets with a resolution better than 1.5 A from crystals glued to glass fibers. Such reinforcement of the crystals facilitates their handling at ambient conditions and opens new possibilities for the measurement of physical properties of large biological macromolecules as well as for their technological applications.

Synthesis of a new hydroxyapatite-silica composite material

Abstract Hydroxyapatite was synthesised by diffusion of calcium ions throughout an alkaline silica gel matrix. Pure hydroxyapatite was obtained when the initial pH value of the gel was set in the range between 10 and 12. The crystals were analysed by X-ray diffraction, scanning electron microscopy, high-resolution transmission electron microscopy and infrared spectroscopy. After drying, a solid composite material is obtained, formed by a nanometer-scale hydroxyapatite crystalline phase embedded into an amorphous silica matrix.

Crystallization and cryocrystallography inside X-ray capillaries

This paper presents a modification of the gel acupuncture method to grow isolated crystals inside X-ray capillaries. Protein crystals are grown from 2–12 µl of gelled agarose–protein solution, cryoprotected and immobilized by the gel matrix. The same X-ray capillary that acts as a crystallization reactor is used to transport the crystals to the X-ray source and to collect data at both room temperature and 100 K, without any post-crystallization manipulation. To enhance the flash-cooling stage, two additional elements are proposed for inclusion in the cryosystems currently in use: a laser pointer to illuminate the crystal to be flash-cooled and a trap to divert the N2 flow and switch from room temperature to 100 K without misalignment of the crystal. With the proposed implementation, data can be collected at different temperatures from the same crystal in exactly the same orientation. This permits the study, at lattice level, of changes in unit-cell parameters, mosaic spread and crystal quality induced by cryogenic temperatures and annealing techniques.

Crystal quality of lysozyme single crystals grown by the gel acupuncture method

Abstract Lysozyme single crystals up to 2.0 mm in size have been grown from solution into capillaries which are previously punctuated into a gel layer through which the precipitant agent diffuse. These large single crystals show an excellent optical quality and it is demonstrated by in situ X-ray diffraction that they diffract up to 1.8–1.9 A.

Structure of dihydropyrimidinase from Sinorhizobium meliloti CECT4114: new features in an amidohydrolase family member.

The recombinant dihydropyrimidinase from Sinorhizobium meliloti CECT4114 (SmelDhp) has been characterised and its crystal structure elucidated at 1.85A. The global architecture of the protein is reminiscent of that of the amidohydrolase superfamily, consisting of two domains; an (alpha/beta)(8) TIM-like barrel domain, where the catalytic centre is located, and a smaller beta-sheet sandwich domain of unknown function. The c-terminal tails of each subunit extend toward another monomer in a swapping-like manner, creating a hydrogen bond network which suggests its implication in protein oligomerisation. Mutational and structural evidence suggest the involvement of a conserved tyrosine in the reaction mechanism of the enzyme. SmelDhp presents both hydantoinase and dihydropyrimidinase activities, with higher affinity for the natural six-membered ring substrates. For the five-membered ring substrates, affinity was greater for those with aliphatic and apolar groups in the 5th carbon atom, with the highest rates of hydrolysis for d-5-methyl and d-5-ethyl hydantoin (k(cat)/K(m)=2736+/-380 and 944+/-52M(-1)s(-1), respectively). The optimal conditions for the enzyme activity were found to be 60 degrees C of temperature at pH 8.0. SmelDhp retains 95% of its activity after 6-hour preincubation at 60 degrees C. This is the first dihydropyrimidinase used for the hydrolytic opening of non-natural 6-monosubstituted dihydrouracils, which may be exploited for the production of beta-amino acids.

Growth of shaped single crystals of proteins

We present a procedure for obtaining protein single crystals that fill the capillary tubes in which they grow. The implementation was typical of the gel acupuncture method and the four different proteins are used as examples: lysozyme (HEW), thaumatin I, ferritin and insulin. Rod- and prismatic-shaped protein single crystals of these four proteins were grown inside capillary tubes of 0.2, 0.3, 0.5 mm in diameter and, for the case of lysozyme, up to 1.2 mm in diameter. The maximum length measured along the long axes of the rod crystals was 1.6 mm again for lysozyme crystals. It was observed that, once the capillary tube was filled, the crystal continues to grow by diffusion of the precipitating agent throughout the porous network formed by the protein crystal structure. We also discuss the possibility of growing these cylinders of crystalline proteins by the addition of protein solution to the mother liquor through the upper end of the glass capillary while the precipitating agent diffuses through the protein crystal itself. X-ray diffraction patterns confirm the single crystal character of the protein rods.

Structural Study of the Type II 3-Dehydroquinate Dehydratase from Actinobacillus Pleuropneumoniae

The structure of the type II dehydroquinate dehydratase (DHQase) from Actinobacillus pleuropneumoniae, the third enzyme of the shikimate pathway, has been determined. Crystals diffracting to 1.7 A were obtained in space and on earth using the counter-diffusion technique. The structure was solved using molecular replacement and refined to high resolution. The overall structure of the dodecameric enzyme is described and compared with structures of DHQases from other bacteria. DHQases contain a flexible loop that presumably closes over the active site upon substrate binding. The enzyme can exist in an open or closed conformation. The present structure displays the open conformation, with a sulfate anion bound in the active site. The availability of this structure opens a route to structure-based antibiotics targetting this pathogenic bacterium.

Soaking: the effect of osmotic shock on tetragonal lysozyme crystals

Protein crystals crack when they are soaked in a solution with ionic strength sufficiently different from the environment in which they grew. It is demonstrated for the case of tetragonal lysozyme that the forces involved and the mechanisms that lead to the formation of cracks are different for hypertonic and hypotonic soaking. Tetragonal lysozyme crystals are very sensitive to hypotonic shocks and, after a certain waiting time, cracks always appear with a characteristic pattern perpendicular to the crystallographic c axis. Conversely, a hypertonic shock is better withstood: cracks do not display any deterministic pattern, are only visible at higher differences in ionic strength and after a certain time a phenomenon of crystal reconstruction occurs and the cracks vanish. At the lattice level, the unit-cell volume expands in hypotonic shock and shrinks under hypertonic conditions. However, the compression of the unit cell is anisotropic: the c axis is compressed to a minimum, beyond which it expands despite the unit-cell volume continuing to shrink. This behaviour is a direct consequence of the positive charge that the crystals bear and the existence of channels along the crystallographic c axis. Both features are responsible for the Gibbs-Donnan effect which limits the free exchange of ions and affects the movement of water inside the channels and bound to the protein.