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FP6

BIOCRYSTALLOGENESIS Informe resumido

Project ID: 4898
Financiado con arreglo a: FP6-MOBILITY
País: France

Final Activity Report Summary - BIOCRYSTALLOGENESIS (Biocrystallogenesis of RNA - protein systems)

X-ray crystallography is a major investigation tool in structural biology with many applications in basic research, structural genomics and drug design. It gives access to the three-dimensional picture - or 3D structure - of biomolecules and provides a wealth of information about molecular architecture and recognition. Biocrystallography is widely used to study interactions between proteins and nucleic acids (DNA or RNA), big cellular complexes and nanomachines made of dozens of macromolecules, and the binding of ligands in the active sites of biological catalysts called enzymes. To be applicable, individual cellular components or complexes need to be crystallised. Since ever, the search for crystallisation conditions has been empirical because based on trial-and-error screening of a great variety of chemicals and mixtures. As a consequence, there is a strong demand for rational methods that accelerate the finding of appropriate conditions, facilitate the preparation of crystals and the optimization of their quality.

The present Marie Curie ERG project focused on practical aspects of biological crystal growth with two main objectives: first, the development of novel crystallisation strategies, in particular for improving crystal quality, and second, their validation and their application to the structure determination of aminoacyl-tRNA synthetases and related proteins studied in the host laboratory.

Novel crystallisation strategies:
The effect of magnetic field on protein crystallisation was first investigated with two bacterial enzymes involved in the protein synthesis process, aspartyl- and glutaminyl-tRNA synthetases (AspRS and GlnRS, respectively). The first tests show that GlnRS crystals get oriented in the magnetic field. Whether magnetic fields enhance crystal quality remains an open question but they have clearly an effect.

A comparative analysis of AspRS crystals obtained in different conditions confirmed that growth in agarose gel or in capillaries yields better results. Former studies on small proteins demonstrated that crystals produced in diffusive environments (microgravity, capillary tubes or gels) display lesser defects and enhanced diffraction. In particular, the characterisation of a gel-grown AspRS crystal soaked with a substrate analog yielded one of the best structural data ever collected for this protein, leading to the unambiguous visualisation of the analog in its active site.

Miniaturisation of crystallisation methods recently led to the design of the first microfluidic chip for protein crystallisation. In 2004, we set up a collaboration with two teams of physicists to develop a novel and versatile microfluidic chip dedicated to screening, crystallisation optimisation and in situ crystal analysis by X-ray diffraction. The feasibility of the concept has been demonstrated and a patent application has been deposited.

Examples of applications to structural studies:
The characterisation of GlnRS combining biochemical and structural approaches showed that this enzyme in the bacterium D. radiodurans possesses a unique C-terminal extension which appears to be mobile in the 3D structure. When this extension is deleted, the binding affinity of GlnRS for its main substrate, the tRNA, is strongly reduced.

Crystals of AspRS, GlnRS and related enzymes were soaked in solutions containing their natural substrates (amino acids, ATP) or chemical analogs. 3D structures revealed that substrate binding was often accompanied by conformational changes in the neighborhood of the catalytic cleft. As a consequence, we now systematically introduce small substrates as 'structure stabilisers' when enzymes are reluctant to crystallise.

Contacto

Richard GIEGE, (Research Director)
Tel.: +33388417058
Fax: +33388602218
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