Vous êtes ici : Accueil > L'institut > Crystallization of membrane proteins with an automated microfluidic pipeline


Soutenance de thèse

Crystallization of membrane proteins with an automated microfluidic pipeline

Mardi 08 juin 2021 à 14:00, Visioconférence

Publié le 8 juin 2021
Sofia Jaho
Institut de Biologie Structurale, Institut de Recherche Interdisciplinaire de Grenoble
Instrumental and methodological developments were implemented to address some of the challenges encountered in protein crystallography. We have developed a microfluidic chip and a platform (MicroCrys) for optimizing and rationalizing protein crystallization. Model soluble and membrane proteins were crystallized on chip with the microdialysis method and the crystals were used for in situ synchrotron serial X-ray crystallography (SSX). Microdialysis, combined with temperature control,offers a reversible and precise control over crystallization and can be used to decouple nucleation from crystal growth for investigating protein phase diagrams. Automating the on chip crystallization process with MicroCrys allows crystallization solution to be dynamically exchanged within the microfluidic channel in a continuous circulation mode, without wasting the protein sample. Thus, crystallization conditions can be screened and phase diagrams can be explored using the dialysis chip. Custom-built computer software provides a user-friendly graphical interface to manipulate the visualization, the fluidic and the thermal components of MicroCrys. Moreover, the lipidic cubic phase (LCP) method for protein crystallization was used on chip in combination with microdialysis. Numerous, isomorphous and well-diffracting micro-crystals of soluble proteins grew on chip applying the LCP approach. Further studies were conducted to strengthen the direction of our developments and to validate them. The background noise generated by the materials comprising our microfluidic device was minimized rendering the chips compatibility with in situ diffraction data collection. The current microchip uses a RC membrane,integrated within two layers of the device, for on chip microdialysis crystallization. However, the novel approach of fabricating hydrogel membranes directly on microchips via in situ photo-polymerization was considered and such a chip was designed for preliminary tests. Finally, the effect of radiation damage on the protein crystals during in situ X-ray diffraction experiments at room temperature was investigated in order to evaluate and propose data collection strategies for future SSX experiments.

Pour suivre la soutenance en visioconférence : https://cnrs.zoom.us/j/91344443744

ID de réunion : 913 4444 3744
code : 2uU0BR