Abstract
At the foundation of many cellular processes as well as a large number of diseases is the (mis)folding of important intrinsically disordered proteins (IDPs). Despite tremendous scientific efforts, the factors driving their structural changes within the cellular context remain poorly understood. In-cell NMR spectroscopy enables investigation of IDPs directly in the living eukaryotic cell enabling investigation of its intermolecular interactions and ensuing modifications at an unprecedented atomic resolution. In the following protocol, we describe how to prepare in-cell NMR samples of IDPs within eukaryotic cells and how to measure these in-cell NMR samples of an IDP in its natural environment, the living mammalian cell. Furthermore, we outline a procedure to assess the intracellular recombinant protein concentration of the studied IDP based on in-cell NMR methods. We use α-synuclein as a model protein, but the presented approach is highly modular and therefore should be easily adapted and altered to the desired needs for the studies of different IDPs.
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Acknowledgments
B.M.B. gratefully acknowledges funding from the Swedish Research Council and the Knut och Alice Wallenberg Foundation through a Wallenberg Academy Fellowship as well as through the Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Sweden. The Swedish NMR Centre of the University of Gothenburg is acknowledged for spectrometer time.
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Matečko-Burmann, I., Burmann, B.M. (2020). Recording In-Cell NMR-Spectra in Living Mammalian Cells. In: Kragelund, B.B., Skriver, K. (eds) Intrinsically Disordered Proteins. Methods in Molecular Biology, vol 2141. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0524-0_44
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DOI: https://doi.org/10.1007/978-1-0716-0524-0_44
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