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In-cell 13C NMR spectroscopy for the study of intrinsically disordered proteins

Nature Protocols volume 9, pages 2005–2016 (2014)Cite this article

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Abstract

A large number of proteins carry out their function in highly flexible and disordered states, lacking a well-defined 3D structure. These proteins, referred to as intrinsically disordered proteins (IDPs), are now in the spotlight of modern structural biology. Nuclear magnetic resonance (NMR) spectroscopy represents a unique tool for accessing atomic resolution information on IDPs in complex environments as whole cells, provided that the methods are optimized to their peculiar properties and to the characteristics of in-cell experiments. We describe procedures for the preparation of in-cell NMR samples, as well as for the setup of NMR experiments and their application to in-cell studies, using human α-synuclein overexpressed in Escherichia coli as an example. The expressed protein is labeled with 13C and 15N stable isotopes to enable the direct recording of 13C-detected NMR experiments optimized for the properties of IDPs. The entire procedure covers 24 h, including cell transformation, cell growth overnight, setup of the spectrometer and NMR experiment recording.

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Figure 1: Analysis of IDPs at atomic resolution.
Figure 2: Schematic representation of the 2D spectra that can be useful to study in-cell IDPs.
Figure 3: Correlation spectra of α-synuclein overexpressed in E. coli cells and on cell lysates.
Figure 4: The 2D CON spectra acquired on α-synuclein overexpressed in E. coli cells using two different variants of the experiment.
Figure 5: Selected regions of the 2D 1H-15N (2D HN SOFAST) and 2D 13C-15N (2D HN-BESTCON and 2D Hα-flipCON) correlation spectra.
Figure 6: 2D 13C′-13Cα correlation spectra (2D CACO).

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Acknowledgements

This work has been supported in part by the European Commission Projects INSTRUCT (contract no. 211252), BioNMR (contract no. 261863) and IDPbyNMR (contract no. 264257).

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Authors and Affiliations

  1. Department of Chemistry 'Ugo Schiff', Magnetic Resonance Center (CERM), University of Florence, Sesto Fiorentino, Italy

    Isabella C Felli, Leonardo Gonnelli & Roberta Pierattelli

Authors
  1. Isabella C Felli
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  2. Leonardo Gonnelli
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  3. Roberta Pierattelli
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Contributions

I.C.F. and R.P. conceived the protocol, designed and performed NMR experiments, analyzed data and wrote the paper; L.G. designed, performed and wrote the procedure for the preparation of the samples.

Corresponding authors

Correspondence to Isabella C Felli or Roberta Pierattelli.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Data 1

Variants for the acquisition of 2D 13C'-15N correlation experiments. (PDF 435 kb)

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Felli, I., Gonnelli, L. & Pierattelli, R. In-cell 13C NMR spectroscopy for the study of intrinsically disordered proteins. Nat Protoc 9, 2005–2016 (2014). https://doi.org/10.1038/nprot.2014.124

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