Macromolecular Crystallization Controlled by Colloidal Interactions: The Case of Urate Oxidase

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Coupling high throughput microfluidics and small-angle x-ray scattering to study protein crystallization from solution

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

Innovative high-throughput SAXS methodologies based on photonic lab-on-a-chip sensors: application to macromolecular studies

The relevance of coupling droplet-based Photonic Lab-on-a-Chip (PhLoC) platforms and Small-Angle X-Ray Scattering (SAXS) technique is here highlighted for the performance of high throughput investigations, related to the study of protein macromolecular interactions. With this configuration, minute amounts of sample are required to obtain reliable statistical data. The PhLoC platforms presented in this work are designed to allow and control an effective mixing of precise amounts of proteins, crystallization reagents and buffer in nanoliter volumes, and the subsequent generation of nanodroplets by means of a two-phase flow. Spectrophotometric sensing permits a fine control on droplet generation frequency and stability as well as on concentration conditions, and finally the droplet flow is synchronized to perform synchrotron radiation SAXS measurements in individual droplets (each one acting as an isolated microreactor) to probe protein interactions. With this configuration, droplet physic-chemical conditions can be reproducibly and finely tuned, and monitored without cross-contamination, allowing for the screening of a substantial number of saturation conditions with a small amount of biological material. The setup was tested and validated using lysozyme as a model of study. By means of SAXS experiments, the proteins gyration radius and structure envelope were calculated as a function of protein concentration. The obtained values were found to be in good agreement with previously reported data, but with a dramatic reduction of sample volume requirements compared to studies reported in the literature

Quand les interactions faibles contrôlent les diagrammes de phases: application a une purification industrielle par cristallisation.

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Quand les interactions faibles contrôlent les diagrammes de phases.

International audienc

Knowledge of weak interactions between proteins in solution for the development of a purification process by crystallization.

International audienc

Quand les interactions faibles contrôlent les diagrammes de phases.

International audienc

Analysis and modeling of SDS and DPC micelle SAXS data for membrane protein solution structure characterization

Herein, we present analysis and analytical modeling of Small Angle X-ray Scattering (SAXS) data on two surfactants forming micelles (i.e., sodium dodecyl sulfate and dodecyl phosphocholine) and used for the study in solution of mTSPO, the translocator membrane protein from Mus musculus, as supporting data of the research article published in Biochimie (Combet et al., 2022).For both surfactants, concentration series were measured at two Synchrotron SAXS-beamlines. After reduction, buffer subtraction and water calibration of the data, SAXS curves were normalized to surfactant concentration to highlight possible changes in micelle shape or presence of inter-micellar weak interactions. They were then analyzed in terms of radius of gyration (RG), absolute forward intensity (I0) to access the surfactant aggregation number (Na) and pair-distance distribution function (P(r)), which gives information on the shape and dimensions of the micelles. Finally, an analytical modeling using SasView - a SAS analysis software package (https://www.sasview.org/) - was performed to describe structural features of the two surfactant micelles at a concentration at which no change in the micelle shape nor weak interactions are observed. A core-shell ellipsoidal model was used to fit the SAXS curves, which provided geometrical parameters of the micelles (equatorial and polar radii, shell thickness) and also scattering length densities (SLD) of both the hydrophobic core and the hydrophilic shell. Hydration of polar heads into the micelle shell could be estimated from micelle volume calculations (Vcore and Vshell). These parameters are particularly useful when modeling SAXS curves of membrane protein-surfactant complexes as described in Combet et al. (2022)

Properties of macromolecular solutions relevant to crystallization: Use of new amphiphilic molecules for soluble and membrane protein crystallization.

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Properties of macromolecular solutions relevant to crystallization: Use of new amphiphilic molecules for soluble and membrane protein crystallization.

International audienc