Jose A. Gavira

Protein crystallization by capillary counterdiffusion for applied crystallographic structure determination

Counterdiffusion crystallization in capillary is a very simple, cost-effective, and practical procedure for obtaining protein crystals suitable for X-ray data analysis. Its principles have been derived using well-known concepts coupling the ideas of precipitation and diffusion mass transport in a restricted geometry. The counterdiffusion process has been used to simultaneously screen for optimal conditions for protein crystal growth, incorporate strong anomalous scattering atoms, and mix in cryogenic solutions in a single capillary tube. The crystals obtained in the capillary have been used in situ for X-ray analysis. The implementation of this technique linked to the advancement of current crystallography software leads to a powerful structure determination method consolidating crystal growth, X-ray data collection, and ab initio phase determination into one without crystal manipulation. We review the historical progress of counterdiffusion crystallization, its application to X-ray crystallography, and ongoing tool development for high-throughput protein structure determination.

Hyperstability and Substrate Promiscuity in Laboratory Resurrections of Precambrian β‑Lactamases

We report a sequence reconstruction analysis targeting several Precambrian nodes in the evolution of class-A β-lactamases and the preparation and experimental characterization of their encoded proteins. Despite extensive sequence differences with the modern enzymes (~100 amino acid differences), the proteins resurrected in the laboratory properly fold into the canonical lactamase structure. The encoded proteins from 2-3 billion years (Gyr)-old β-lactamase sequences undergo cooperative two-state thermal denaturation and display very large denaturation temperature enhancements (~35 °C) relative to modern β-lactamases. They degrade different antibiotics in vitro with catalytic efficiencies comparable to that of an average modern enzyme. This enhanced substrate promiscuity is not accompanied by significant changes in the active-site region as seen in static X-ray structures, suggesting a plausible role for dynamics in the evolution of function in these proteins. Laboratory resurrections of 2-3 Gyr-old β-lactamases also endowed modern microorganisms with significant levels of resistance toward a variety of antibiotics, opening up the possibility of performing laboratory replays of the molecular tape of lactamase evolution. Overall, these results support the notions that Precambrian life was thermophilic and that proteins can evolve from substrate-promiscuous generalists into specialists during the course of natural evolution. They also highlight the biotechnological potential of laboratory resurrection of Precambrian proteins, as both high stability and enhanced promiscuity (likely contributors to high evolvability) are advantageous features in protein scaffolds for molecular design and laboratory evolution.

Counterdiffusion methods applied to protein crystallization.

Accumulated experience during the last years on counterdiffusion crystallization methods shows that they are a convenient and generally applicable way of optimizing solution crystal growth experiments. Irrespective of whether the objective of the experiment is to improve crystal quality or size, many experiments reporting a positive or neutral effect of counterdiffusion exists, but adverse effects are consistently absent. Thus counterdiffusion is viewed as a rational crystallization approach to minimize supersaturation and impurity levels at the crystal growth front and to ensure steadiness of both values. This control of the phase transition state is automatically achieved and sustained by a dynamic equilibrium between mass transport and aggregation kinetics. The course of this function can be implemented in any media permitting diffusive mass transport (gels, capillaries, microfluidic devices or microgravity). The counterdiffusion technique has been exploited in many recent applications revealing interesting effects on nucleation and polymorphic precipitation, hence opening further possibilities for innovative screening of crystallization conditions.

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.

Introduction to protein crystallization

Protein crystallization was discovered by chance about 150 years ago and was developed in the late 19th century as a powerful purification tool and as a demonstration of chemical purity. The crystallization of proteins, nucleic acids and large biological complexes, such as viruses, depends on the creation of a solution that is supersaturated in the macromolecule but exhibits conditions that do not significantly perturb its natural state. Supersaturation is produced through the addition of mild precipitating agents such as neutral salts or polymers, and by the manipulation of various parameters that include temperature, ionic strength and pH. Also important in the crystallization process are factors that can affect the structural state of the macromolecule, such as metal ions, inhibitors, cofactors or other conventional small molecules. A variety of approaches have been developed that combine the spectrum of factors that effect and promote crystallization, and among the most widely used are vapor diffusion, dialysis, batch and liquid-liquid diffusion. Successes in macromolecular crystallization have multiplied rapidly in recent years owing to the advent of practical, easy-to-use screening kits and the application of laboratory robotics. A brief review will be given here of the most popular methods, some guiding principles and an overview of current technologies.

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.

Agarose as crystallisation media for proteins II: Trapping of gel fibres into the crystals

The crystallisation pressure exerted by protein crystals growing in agarose gel does not disrupt the gel network. However, protein crystals trap agarose fibres when they grow in agarose gels. The fibres of agarose are distributed randomly in the crystals explaining why they do not appreciably affect the diffraction quality of the crystal

Topography and high resolution diffraction studies in tetragonal lysozyme

Abstract Two complementary approaches are used to enhance the usefulness of X-ray topographies obtained from protein crystals. First, the use of thin plate-like crystals in conjunction with a high intensity, collimated and small source size synchrotron beam produces a large beneficial effect on the level of detail and contrast of topographies for the quantification of local misalignment in the crystal lattice. Second, the recording of topography series along the rocking curve of a diffraction peak is proposed as a technique to combine the benefits of both rocking curves and topographies and produce very detailed data (“rocking maps”) on the spatial distribution of lattice misalignments and mosaic spread (“local rocking curves”). The most important crystal features controlling the observed contrast are growth sectors and inter-sector boundaries, clearly observed in the topographies. Systems of parallel fringes are observed in many of the topographies. Two alternative explanations for these fringes are discussed: (a) as moire interference fringes or (b) as Pendellosung fringes in a wedge shaped crystal volume. In both cases, growth sectors play a central role in the physics of fringe generation. Many observations suggest the presence of a relatively large component of dynamical diffraction in these crystals; the consequences of this new scenario are discussed.

Ab initio crystallographic structure determination of insulin from protein to electron density without crystal handling

Insulin crystals suitable for cryogenic data collection and structure determination by single-wavelength anomalous scattering (SAS) were obtained by a self-optimization screening process in a single capillary tube without manipulation of the crystals at any time. Using the counter-diffusion crystallization technique, screening for optimal conditions for crystal growth, incorporation of a strong anomalous scattering halide and cryogenic solution took place simultaneously in a single capillary tube. The crystals in the capillaries can be placed directly in the cryostream for data collection using a conventional home-laboratory X-ray source. High-redundancy data were used to obtain a Patterson solution from the anomalous signal of iodine. As a result, the anomalous scattering-atom position was determined and the phase calculated, giving rise to an initial electron-density map at 2.4 A resolution. This entire procedure from crystal growth to the determination of an initial structure was performed within four weeks.