Cristalización de macromoléculas biológicas en condiciones terrestres y de microgravedadla técnica de contradifusión

Resumen

Proteins are biological macromolecules which carry out a broad variety of key functions in living organisms. Practically every cellular activity depends on the actions of these molecules. The function that these biomacromolecules perform is intimately related to their tridimensional atomic structures. By determining this atomic structure it is possible to understand the mechanism of these functions. Up to now the most effective technique to determine a tridimensional structure of biological macromolecules is X-ray diffraction. However, the essential requirement to apply this technique is the availability of high quality single crystals from these macromolecules. The counterdiffusion technique is a crystallization technique that in comparison to the classic techniques has been shown to produce quality crystals for X-ray studies. This thesis focused mainly on the features (or characteristics) of this technique and the development of practical devices for its implementation in research laboratories. The Granada Crystallization Box (GCB) is a new crystallization device designed to perform counter-diffusion experiments. In chapter two the device and its use for protein crystallization purposes is described. GCB allows one to explore and exploit the coupling between crystallization 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, which is known to produce better ordered crystal lattices, provided the growth proceeds in the diffusion-controlled or mixed regime. The use of capillaries also reduces the consumption of macromolecules and avoids the handling of crystals for X-ray diffraction data collection. The use of crystallization methods in which the mass transport is controlled by diffusion is one of the ways to improve the quality of crystals of biological macromolecules for structural studies. Moreover, a fluid dynamics governed by diffusion is mandatory for some crystallization techniques as well as for the counterdiffusion technique. The use of gels is extremely effective in the elimination of convection generated by thermal and density fluctuations, in this way allowing diffusion to govern the mass and heat transport in crystallization reactors. The main drawback in the utilization of these gels is that they are chemical compounds that could interact with the biological macromolecules and with the different chemical reagents used to crystallize them. In chapter three, a study of compatibility between different types of gels (thermal and physical gels) and precipitating agents, additives and detergents commonly used for protein crystallization experiments is described. The physical and chemical stability as a function of the pH value of the different buffers used in the experiments was also investigated. Different types of gels were analyzed, namely, silica gels, agarose gels, and polyacrylamide gels. The compatibility of these compounds was studied in the presence of the components of the Crystal Screen I kit from Hampton Research under typical conditions used in crystallization experiments. For this kind of study the Detergent Screen I from the same company, which is commonly used for crystallization of membrane proteins, was also included. The results showed that all the detergents commonly used in the crystallization of membrane proteins were compatible with all the types of gels analyzed. Regarding the precipitating agents, it was observed that some of them, such as ammonium sulfate and high molecular weight polyethylenglycoles, inhibited the formation of agarose gels. However, agarose gels were able to form in all the pH values analyzed, this not being the case for silica gels which were more sensitive to this parameter. It is showed that, in view of the obtained results, it is possible to carry out experiments of crystallization of biological macromolecules under mass transport environment controlled by diffusion through the use of gels as crystal growth media. Although some chemical reagents inhibited the formation of gels, there are still alternative combinations with other gels that did not show incompatibility. Chapter Four details two interlinked investigations: firstly, examining the viability of carrying out experiments of crystallization of biological macromolecules in microgravity conditions; and, secondly, it was proposed to make a comparison between the quality of crystals obtained in two different diffusive environments: one in which the crystals grew on earth with gels as crystallization media and the other in outer space with a reduced gravity force environment. The results from three different space missions supported by the European Space Agency (ESA) are described, two carried out inside the International Space Station (Andromede and Odissea Missions) and one inside a Russian space capsule (Foton M-3 Mission). For the three missions it was necessary to design and fabricate special facilities to contain the crystallization experiments during the missions, namely the Granada Crystallization Facility (GCF) and its improved version, the GCF-2, for the Foton M-3 Mission. GCF-2 is an inexpensive and compact modular platform featuring a high experiment density (number of experiments per litre of useful volume), a temperature control system, and a complete independence from human intervention after integration, which makes GCF-2 equally suited for manned and unmanned flights. The facility can be used for commercial quality crystal production and for fundamental crystal growth studies using ex-situ diagnostics. The changes made to the previous version and the complementary hardware (electronics and reactors) used are described. Also included is information on the performance of the facility during the Foton-M3 mission on board the Russian unmanned Foton capsule. In all the missions described in this chapter, different proteins provided by different European laboratories and by our own laboratory were tested. The quality of the crystals was analysed through X-ray diffraction in Synchrotron facilities. The results showed that all the devices designed and fabricated ad hoc for the different missions worked in a satisfactory way. The results obtained of crystal quality did not show significant differences between the crystals grown in space and the crystals grown in gellified solutions on earth.