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Open AccessArticle

Multi-Step Concanavalin A Phase Separation and Early-Stage Nucleation Monitored Via Dynamic and Depolarized Light Scattering

1
Center for Free-Electron Laser Science (CFEL), Deutsches Elektronen-Synchrotron (DESY), 22607 Hamburg, Germany
2
Institute of Biochemistry and Molecular Biology, Laboratory for Structural Biology of Infection and Inflammation, University of Hamburg, c/o DESY. Build. 22a, Notkestrasse 85, 22607 Hamburg, Germany
3
The Hamburg Centre for Ultrafast Imaging, 22607 Hamburg, Germany
*
Author to whom correspondence should be addressed.
Crystals 2019, 9(12), 620; https://doi.org/10.3390/cryst9120620
Received: 5 September 2019 / Revised: 20 November 2019 / Accepted: 22 November 2019 / Published: 26 November 2019
Protein phase separation and protein liquid cluster formation have been observed and analysed in protein crystallization experiments and, in recent years, have been reported more frequently, especially in studies related to membraneless organelles and protein cluster formation in cells. A detailed understanding about the phase separation process preceding liquid dense cluster formation will elucidate what has, so far, been poorly understood—despite intracellular crowding and phase separation being very common processes—and will also provide more insights into the early events of in vitro protein crystallization. In this context, the phase separation and crystallization kinetics of concanavalin A were analysed in detail, which applies simultaneous dynamic light scattering and depolarized dynamic light scattering to obtain insights into metastable intermediate states between the soluble phase and the crystalline form. A multi-step mechanism was identified for ConA phase separation, according to the resultant ACF decay, acquired after an increase in the concentration of the crowding agent until a metastable ConA gel intermediate between the soluble and final crystalline phases was observed. The obtained results also revealed that ConA is trapped in a macromolecular network due to short-range intermolecular protein interactions and is unable to transform back into a non-ergodic solution. View Full-Text
Keywords: liquid dense cluster; liquid–liquid phase separation; gelation; dynamic light scattering; depolarized dynamic light scattering; nucleation; carbohydrate-binding protein; response surface methodology liquid dense cluster; liquid–liquid phase separation; gelation; dynamic light scattering; depolarized dynamic light scattering; nucleation; carbohydrate-binding protein; response surface methodology
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MDPI and ACS Style

Brognaro, H.; Falke, S.; Nzanzu Mudogo, C.; Betzel, C. Multi-Step Concanavalin A Phase Separation and Early-Stage Nucleation Monitored Via Dynamic and Depolarized Light Scattering. Crystals 2019, 9, 620.

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