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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References
Babu MM, van der Lee R, de Groot NS, Gsponer J (2011) Intrinsically disordered proteins: regulation and disease. Curr Opin Struct Biol 21(3):432–440. https://doi.org/10.1016/j.sbi.2011.03.011
Snead D, Eliezer D (2019) Intrinsically disordered proteins in synaptic vesicle trafficking and release. J Biol Chem 294(10):3325–3342. https://doi.org/10.1074/jbc.REV118.006493
Kadavath H, Jaremko M, Jaremko L, Biernat J, Mandelkow E, Zweckstetter M (2015) Folding of the Tau protein on microtubules. Angew Chem Int Ed Engl 54(35):10347–10351. https://doi.org/10.1002/anie.201501714
Dunker AK, Oldfield CJ, Meng J, Romero P, Yang JY, Chen JW, Vacic V, Obradovic Z, Uversky VN (2008) The unfoldomics decade: an update on intrinsically disordered proteins. BMC Genomics 9(Suppl 2):S1. https://doi.org/10.1186/1471-2164-9-S2-S1
Dunker AK, Silman I, Uversky VN, Sussman JL (2008) Function and structure of inherently disordered proteins. Curr Opin Struct Biol 18(6):756–764. https://doi.org/10.1016/j.sbi.2008.10.002
Uversky VN, Oldfield CJ, Dunker AK (2008) Intrinsically disordered proteins in human diseases: introducing the D2 concept. Annu Rev Biophys 37:215–246. https://doi.org/10.1146/annurev.biophys.37.032807.125924
Wright PE, Dyson HJ (2015) Intrinsically disordered proteins in cellular signalling and regulation. Nat Rev Mol Cell Biol 16(1):18–29. https://doi.org/10.1038/nrm3920
Gsponer J, Futschik ME, Teichmann SA, Babu MM (2008) Tight regulation of unstructured proteins: from transcript synthesis to protein degradation. Science 322(5906):1365–1368. https://doi.org/10.1126/science.1163581
Goedert M, Spillantini MG, Del Tredici K, Braak H (2013) 100 Years of Lewy pathology. Nat Rev Neurol 9(1):13–24. https://doi.org/10.1038/nrneurol.2012.242
Lashuel HA, Overk CR, Oueslati A, Masliah E (2013) The many faces of α-Synuclein: from structure and toxicity to therapeutic target. Nat Rev Neurosci 14(1):38–48. https://doi.org/10.1038/nrn3406
Selenko P, Serber Z, Gadea B, Ruderman J, Wagner G (2006) Quantitative NMR analysis of the protein GB1 domain in Xenopus laevis egg extracts and intact oocytes. Proc Natl Acad Sci U S A 103(32):11904–11909. https://doi.org/10.1073/pnas.0604667103
Serber Z, Selenko P, Hansel R, Reckel S, Löhr F, Ferrell JE Jr, Wagner G, Dötsch V (2006) Investigating macromolecules inside cultured and injected cells by in-cell NMR spectroscopy. Nat Protoc 1(6):2701–2709. https://doi.org/10.1038/nprot.2006.181
Barbieri L, Luchinat E, Banci L (2016) Characterization of proteins by in-cell NMR spectroscopy in cultured mammalian cells. Nat Protoc 11(6):1101–1111. https://doi.org/10.1038/nprot.2016.061
Theillet FX, Binolfi A, Bekei B, Martorana A, Rose HM, Stuiver M, Verzini S, Lorenz D, van Rossum M, Goldfarb D, Selenko P (2016) Structural disorder of monomeric α-Synuclein persists in mammalian cells. Nature 530(7588):45–50. https://doi.org/10.1038/nature16531
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Kang L, Moriarty GM, Woods LA, Ashcroft AE, Radford SE, Baum J (2012) N-terminal acetylation of α-synuclein induces increased transient helical propensity and decreased aggregation rates in the intrinsically disordered monomer. Protein Sci 21(7):911–917. https://doi.org/10.1002/pro.2088
Huang C, Ren G, Zhou H, C-c W (2005) A new method for purification of recombinant human α-Synuclein in Escherichia coli. Protein Express Purif 42(1):173–177. https://doi.org/10.1016/j.pep.2005.02.014
Anthis NJ, Clore GM (2013) Sequence-specific determination of protein and peptide concentrations by absorbance at 205 nm. Protein Sci 22(6):851–858. https://doi.org/10.1002/pro.2253
Schanda P, Kupče E, Brutscher B (2005) SOFAST-HMQC experiments for recording two-dimensional heteronuclear correlation spectra of proteins within a few seconds. J Biomol NMR 33(4):199–211. https://doi.org/10.1007/s10858-005-4425-x
Solyom Z, Schwarten M, Geist L, Konrat R, Willbold D, Brutscher B (2013) BEST-TROSY experiments for time-efficient sequential resonance assignment of large disordered proteins. J Biomol NMR 55(4):311–321. https://doi.org/10.1007/s10858-013-9715-0
Bermel W, Bertini I, Duma L, Felli IC, Emsley L, Pierattelli R, Vasos PR (2005) Complete assignment of heteronuclear protein resonances by protonless NMR spectroscopy. Angew Chem Int Ed Engl 44(20):3089–3092. https://doi.org/10.1002/anie.200461794
Gil S, Hosek T, Solyom Z, Kümmerle R, Brutscher B, Pierattelli R, Felli IC (2013) NMR spectroscopic studies of intrinsically disordered proteins at near-physiological conditions. Angew Chem Int Ed Engl 52(45):11808–11812. https://doi.org/10.1002/anie.201304272
Drexler HG, Uphoff CC (2002) Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention. Cytotechnology 39(2):75–90. https://doi.org/10.1023/A:1022913015916
Nikfarjam L, Farzaneh P (2012) Prevention and detection of Mycoplasma contamination in cell culture. Cell J 13(4):203–212
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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