title: Coupling High Throughput Microfluidics and Small-Angle X-ray Scattering to Study Protein Crystallization from Solution.
abstract: In this work, we propose the combination of small-angle X-ray scattering (SAXS) and high throughput, droplet based microfluidics as a powerful tool to investigate macromolecular interactions, directly related to protein solubility. For this purpose, a robust and low cost microfluidic platform was fabricated for achieving the mixing of proteins, crystallization reagents, and buffer in nanoliter volumes and the subsequent generation of nanodroplets by means of a two phase flow. The protein samples are compartmentalized inside droplets, each one acting as an isolated microreactor. Hence their physicochemical conditions (concentration, pH, etc.) can be finely tuned without cross-contamination, allowing the screening of a huge number of saturation conditions with a small amount of biological material. The droplet flow is synchronized with synchrotron radiation SAXS measurements to probe protein interactions while minimizing radiation damage. To this end, the experimental setup was tested with rasburicase (known to be very sensitive to denaturation), proving the structural stability of the protein in the droplets and the absence of radiation damage. Subsequently weak interaction variations as a function of protein saturation was studied for the model protein lysozime. The second virial coefficients (A2) were determined from the X-ray structure factors extrapolated to the origin. A2 obtained values were found to be in good agreement with data previously reported in literature but using only a few milligrams of protein. The experimental results presented here highlight the interest and convenience of using this methodology as a promising and potential candidate for studying protein interactions for the construction of phase diagrams.
authors.affiliation: Laboratoire de Génie Chimique, Université de Toulouse, CNRS, INPT, UPS , 4 allée Emile Monso, 31432 Toulouse, France.
authors.forename: Nhat
authors.initials: N
authors.lastname: Pham
authors.affiliation: Laboratoire de Génie Chimique, Université de Toulouse, CNRS, INPT, UPS , 4 allée Emile Monso, 31432 Toulouse, France.
authors.forename: Dimitri
authors.initials: D
authors.lastname: Radajewski
authors.affiliation: European Molecular Biology Laboratory , 71 avenue des Martyrs, 38042 Grenoble, France.|Unit for Virus Host-Cell Interactions, Université Grenoble Alpes-EMBL-CNRS , 71 avenue des Martyrs, 38042 Grenoble, France.
authors.forename: Adam
authors.initials: A
authors.lastname: Round
authors.affiliation: European Synchrotron Radiation Facility , 71 avenue des Martyrs, 38000 Grenoble, France.
authors.forename: Martha
authors.initials: M
authors.lastname: Brennich
authors.affiliation: European Synchrotron Radiation Facility , 71 avenue des Martyrs, 38000 Grenoble, France.
authors.forename: Petra
authors.initials: P
authors.lastname: Pernot
authors.affiliation: Laboratoire de Génie Chimique, Université de Toulouse, CNRS, INPT, UPS , 4 allée Emile Monso, 31432 Toulouse, France.
authors.forename: Béatrice
authors.initials: B
authors.lastname: Biscans
authors.affiliation: Institut des Biomolécules Max-Mousseron, UMR 5247, Université d'Avignon , 301 rue Baruch de Spinoza, 84000 Avignon, France.
authors.forename: Françoise
authors.initials: F
authors.lastname: Bonneté
authors.affiliation: Laboratoire de Génie Chimique, Université de Toulouse, CNRS, INPT, UPS , 4 allée Emile Monso, 31432 Toulouse, France.
authors.forename: Sébastien
authors.identifier: 0000-0002-4840-1829
authors.initials: S
authors.lastname: Teychené
mesh_terms: D003460:Crystallization; D044085:Microfluidics / Q000379:methods*; D009113:Muramidase / Q000737:chemistry*; D011489:Protein Denaturation; D053838:Scattering, Small Angle; D012995:Solubility; D013501:Surface-Active Agents / Q000737:chemistry; D014503:Urate Oxidase / Q000737:chemistry* / Q000378:metabolism; D014961:X-Ray Diffraction
chemical_list: D013501:Surface-Active Agents; C469709:rasburicase; D014503:Urate Oxidase; D009113:Muramidase