We detail the analysis of centrifugal homogenization process by a hydrodynamic model and the model-guided design of a low-cost centrifugal homogenizer. During operation, centrifugal force pushes a multiphase solution to be homogenized through a thin nozzle, consequently homogenizing its contents. We demonstrate and assess the homogenization of coarse emulsions into relatively monodisperse emulsions, as well as the application of centrifugal homogenization in the mechanical lysis of mpkCCD mouse kidney cells. To gain insight into the homogenization mechanism, we investigate the dependence of emulsion droplet size on geometrical parameters, centrifugal acceleration, and dispersed phase viscosity. Our experimental results are in qualitative agreement with models predicting the droplet size. Furthermore, they indicate that high shear rates kept constant throughout operation produce more monodisperse droplets. We show this ideal homogenization condition can be realized through hydrodynamic model-guided design minimizing transient effects inherent to centrifugal homogenization. Moreover, we achieved power densities comparable to commercial homogenizers by model guided optimization of homogenizer design and experimental conditions. Centrifugal homogenization using the proposed homogenizer design thus offers a low-cost alternative to existing technologies as it is constructed from off-the-shelf parts (Falcon tubes, syringe, needles) and used with a centrifuge, readily available in standard laboratory environment.

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The steep increase of atomic scale structures determined by 3D cryo-electron microscopy (EM) deposited in the EMDataBank documents progress of a methodology that was frustratingly slow ten years ago. While sample vitrification on grids has been successfully used in all EM laboratories for decades, beam damage remains a road block. Developments in instrumentation and software to exploit the information carried by elastically scattered electrons made the task to achieve atomic scale resolution easier. This together with the development of fast single electron detecting cameras has resulted in unprecedented possibilities for structure determination by 3D cryo-EM. With such technologies in place, the purification of membrane protein complexes in a functional state is key to collecting atomic scale structural information and insight into the chemistry of physiological processes. Therefore, we focus here on the preparation of membrane proteins for structural analyses by 3D cryo-EM and the data acquisition of such vitrified samples.

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Challenges in purification and subsequent functionalization of membrane proteins often complicate their biochemical and biophysical characterization. Purification of membrane proteins generally involves replacing the lipids surrounding the protein with detergent molecules, which can affect protein structure and function. Recently, it was shown that styrene–maleic acid copolymers (SMA) can dissolve integral membrane proteins from biological membranes into nanosized discs. Within these nanoparticles, proteins are embedded in a patch of their native lipid bilayer that is stabilized in solution by the amphipathic polymer that wraps the disc like a bracelet. This approach for detergent-free purification of membrane proteins has the potential to greatly simplify purification but does not facilitate conjugation of functional compounds to the membrane proteins. Often, such functionalization involves laborious preparation of protein variants and optimization of labeling procedures to ensure only minimal perturbation of the protein. Here, we present a strategy that circumvents several of these complications through modifying SMA by grafting the polymer with cysteamine. The reaction results in SMA that has solvent-exposed sulfhydrils (SMA-SH) and allows tuning of the coverage with SH groups. Size exclusion chromatography, dynamic light scattering, and transmission electron microscopy demonstrate that SMA-SH dissolves lipid bilayer membranes into lipid nanodiscs, just like SMA. In addition, we demonstrate that, just like SMA, SMA-SH solubilizes proteoliposomes into protein-loaded nanodiscs. We covalently modify SMA-SH-lipid nanodiscs using thiol-reactive derivatives of Alexa Fluor 488 and biotin. Thus, SMA-SH promises to simultaneously tackle challenges in purification and functionalization of membrane proteins.@en