Développements instrumentaux pour le contrôle de la cristallisation par la dialyse : approche microfluidique et analyse aux rayons X

Abstract : Protein crystallization is a key step in elucidating three-dimensional structure of proteins. This very sensitive process depends on many variables that are difficult to control precisely or simultaneously in the existing facilities. Instrumentation developments have concentrated on massive parallel experiments and sample volume reduction used by experiment. With this approach it is relatively easy to find initial crystallization conditions but their optimization to yield well diffracting crystals often proves to be more difficult.The method presented herein differs from the current paradigm, since we propose serial instead of parallel experiments based on the knowledge of phase diagrams. This project is based on a series of developments of instruments used to control and rationalize crystallisation using dialysis method, thus allowing phase diagrams exploration without consuming large quantity of protein sample.This results in a microfluidic device that allows crystallization of proteins by dialysis method, use of a continuous flow of crystallization agent and therefore continuous exchange of crystallization conditions as well as temperature control during experiment. It provides X-rays compatibility for in situ diffraction data collection of crystals grown in the microfluidic chip. This microfluidic system is based on the miniaturization of the crystallization bench which has been improved on electronics for automation, fluid transport to operate at a continuous flow, software development for the control of crystallization parameters, mechanics to improve both dialysis cell and thermoregulation, and finally by the integration of a UV system to perform in situ absorbance measurements that provide the future possibility to measure the solubility of proteins in a dialysis crystallization experiment.Finally both instrumental and methodological developments have been validated by the crystallization of several model proteins whose crystals diffracted succesfully X-rays. Furthermore understanding of the transport of species in solution by dialysis was investigated by combined experimental and theoretical approaches.
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Submitted on : Thursday, March 30, 2017 - 5:47:12 PM
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Niels Junius. Développements instrumentaux pour le contrôle de la cristallisation par la dialyse : approche microfluidique et analyse aux rayons X. Biophysique. Université Grenoble Alpes, 2016. Français. ⟨NNT : 2016GREAY032⟩. ⟨tel-01498968⟩



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