TOPCRYST

Novel tools for crystallisation of macromolecules

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 Obiettivo del progetto (Objective)

'The elucidation of 3-dimensional structures of proteins and other biological macromolecules and complexes is essential for rational drug design, targeting and delivery, biocatalysis, the design of environmentally friendly agrochemicals, the development of biosensors and other nanobiotechnological applications. The most powerful tool for structural analysis is X-ray crystallography, which crucially depends on growth of high diffraction quality crystals. Crystallisation is the least controllable and usually rate-limiting step of the process that goes from cloning a gene to using the structural information for predicting and designing function. TOPCRYST, an academia-industry project, will use Dual Polarimetric Interferometry, pioneered by Farfield Scientific Ltd., to probe crystallisation at its earliest, most crucial stages. This will allow to predict the outcome of crystallisation trials when they are still at their earliest stages and thus to rationally design such experiments in order to lead them to the desired result, i.e. well-diffracting crystals. Transfer of knowledge between academia and industry will tackle the problem of detecting crystal nucleation phenomena at the very earliest stages of crystallisation and holds a number of promises that will be investigated in its course: (i) to guide the choice of pH and buffer, temperature, precipitating agent, additive(s) etc, starting from a limited number of preliminary experiments, thus obviating the need for extensive screening; (ii) to allow to unequivocally distinguish crystalline from amorphous material, something which is not always easy even for an experienced crystalliser, with obvious possibilities of extension to high-throughput environments; (iii) to allow optimisation of conditions under real-time control; (iv) to provide an experimental underpinning to the theoretical understanding of nucleation phenomena (v) to develop novel instrumentation for crystallisation of macromolecules.'

Introduzione (Teaser)

Investigation of molecular structure is critical to the development of pharmaceuticals. A novel optical technique developed by EU-funded scientists tremendously decreases discovery time while enhancing the likelihood of success.

Descrizione progetto (Article)

Information on the majority of protein structures known today has been produced using X-ray crystallography, also called X-ray diffraction. An X-ray beam is directed at solid crystals containing trillions of identical molecules of interest. A computer programme creates a three-dimensional (3D) image from position calculations of every atom in the molecule based on the detected diffraction pattern.

Growing high-quality crystals is a complicated and delicate process that can take months or even years with inferior outcomes in many instances. Scientists exploited a novel technique to detect the quality of crystal nucleation early with EU funding of the project 'Novel tools for crystallisation of macromolecules' (TOPCRYST). Their aim is to enable early detection of protein crystal quality to minimise loss of time, effort and money in the case of poor nucleation.

The dual polarisation interferometry (DPI) technique, developed by TOPCRYST for studying molecular structures and interactions, uses an optical waveguide with light in its core. Any changes in molecule interactions on the waveguide surface are reflected in changes in the light propagation within the waveguide.

Scientists used three molecular system models whose crystallisation conditions are well characterised and are therefore often employed to test new methods. A crystallisation 'signature' or signal pattern occurring only upon successful crystallisation was thus identified and used to define crystallisation conditions for two historically problematic proteins. In addition, a novel crystallisation condition was identified for one of the model proteins.

http://topcryst.chem.demokritos.gr/ (TOPCRYST) thus demonstrated the effectiveness of real-time DPI monitoring in identifying successful crystallisation conditions for macromolecules of interest with significant reduction in time and effort. Increasingly successful structural studies due to this technique are expected to have broad-sweeping impact on drug discovery and health care.