Dynamics of intermediate filament assembly followed in micro-flow by small angle X-ray scattering

Brennich, Martha Elisabeth and Nolting, Jens-Friedrich and Dammann, Christian and Nöding, Bernd and Bauch, Susanne and Herrmann, Harald and Pfohl, Thomas and Köster, Sarah. (2011) Dynamics of intermediate filament assembly followed in micro-flow by small angle X-ray scattering. Lab on a chip, 11 (4). pp. 708-716.

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The assembly of intermediate filaments (IFs) is a complex process that can be recapitulated through a series of distinct steps in vitro. The combination of microfluidics and small angle X-ray scattering (SAXS) provides a powerful tool to investigate the kinetics of this process on the relevant timescales. Microfluidic mixers based on the principle of hydrodynamic focusing allow for precise control of the mixing of proteins and smaller reagents like ions. Here, we present a multi-layer device that prevents proteins from adsorbing to the channel walls by engulfing the protein jet with a fluid layer of buffer. To ensure compatibility with SAXS, the device is fabricated from UV-curable adhesive (NOA 81). To demonstrate the successful prevention of contact between the protein jet and the channel walls we measure the distribution of a fluorescent dye in the device by confocal microscopy at various flow speeds and compare the results to finite element method (FEM) simulations. The prevention of contact enables the investigation of the assembly of IFs in flow by gradually increasing the salt concentration in the protein jet. The diffusion of salt into the jet can be determined by FEM simulations. SAXS data are collected at different positions in the jet, corresponding to different salt concentrations, and they reveal distinct differences between the earliest assembly states. We find that the mean square radius of gyration perpendicular to the filament axis increases from 13 nm(2) to 58 nm(2) upon assembly. Thereby we provide dynamic structural data of a complex assembly process that was amenable up to now only by microscopic techniques.
Faculties and Departments:05 Faculty of Science > Departement Chemie > Former Organization Units Chemistry > Biophysikalische Chemie (Pfohl)
UniBasel Contributors:Pfohl, Thomas
Item Type:Article, refereed
Article Subtype:Research Article
Publisher:Royal Society of Chemistry
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:16 Feb 2017 08:57
Deposited On:14 Sep 2012 07:02

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