European XFEL Seminar | Coupling microfluidics and Small-Angle X-ray scattering to study the whole crystallization process of proteins in solution

by Sébastien Teychené (Laboratoire de Génie Chimique, Toulouse, France)

Wednesday 1 Mar 2017, 08:00 → 09:00 Europe/Berlin

E1.173 (HXQ Schenefeld) (European XFEL)

Small-Angle X-Ray Scattering (SAXS) is a particularly suitable technique to characterize structure and form factors of colloidal systems in solution and therefore to probe nanometer-scale structures. The combination of microfluidics and SAXS provides a powerful tool to investigate phase transitions at different molecular levels and relevant timescales. However, very few studies deal with the combination of SAXS and droplet based microfluidic, and none with application to proteins. In digital microfluidics, the sample is compartmentalized inside droplets, each droplet acting as a microreactor in which the operating conditions (concentration, pH, temperature, etc.) can be finely tuned without cross-contamination. In the framework of a Long Term Project MX-1510 and in a recent collaboration with SIWNG beamline at Soleil synchrotron, we have developed robust droplets based microfluidic experimental set-up to probe protein interactions in solution and phase transitions for nucleation studies. The microfluidic systems can generate droplets containing proteins, crystallization agents and buffer, carried by an immiscible fluorinated oil. A huge number of crystallization conditions can be screened with a small amount of biological material just by changing the flow rates of stock solutions. In the microfluidic platform developed on BM29, the droplets are passed from the chip into the sample holder for exposure to X-ray beam. With this setup, three proteins have been tested: rasburicase, lysozyme and glucose isomerase. The experimental scattering curves of rasburicase obtained in nanoliter droplets fit well with their scattering curves from the crystal structure proving the structural stability of the protein in droplets and the absence of radiation damage. The second in-situ study concerned the variation of lysozyme interactions as a function of salt concentration by continuously changing the relative flow rates of the stock solutions. Finally, the liquid-solid phase transition of glucose isomerase and diffraction peaks of first crystals were observed. In the microfluidic platform developed on SWING the microfluidic chips are directly inserted in the beam. In this setup, tens of droplet are stored in a capillary trap, allowing to follow the temporal evolution of the solution structure inside the droplets. The results obtained in this setup enable to demonstrate the existence of equilibrium clusters prior to crystal nucleation. All these results highlight the advantage of coupling microfluidics and SAXS to study the whole protein crystallization process (i.e. from form factor to crystal nucleation and growth) by finely tuning physico-chemical conditions.