In‐Cell NMR

Abstract

In‐cell NMR refers to the study of biomolecules in living cells by nuclear magnetic resonance (NMR). It provides atomic resolution information of structure, dynamics and interactions of biomolecules in their natural environment that is essential to understand the biological function at the molecular level.

The unique characteristics of the in vivo environment arise from molecular crowding and the effects triggered by cellular activity. Crowding affects differently globular and intrinsically disordered proteins (IDPs). IDPs are privileged targets for in‐cell NMR as these responsive systems are essentially coupled to their environment. Cellular activity causes, among other effects, dynamically evolving posttranslational modifications, often in disordered regions. In‐cell NMR is also a promising tool to study interactions of drugs with biomolecules inside the cell or decorating its external surface that depend on the details of the cellular environment.

Key Concepts

  • In‐cell NMR allows monitoring the structure and dynamics of biomolecules in their native environment.
  • Molecular crowding in the cell cytoplasm affects differently globular and intrinsically disordered proteins.
  • Reversible posttranslational modifications can be individually monitored in real time in living cells or active cell extracts.
  • NMR can study the interaction of ligands with membrane receptors on the cell surface.
  • Matching cell culture stability and NMR sensitivity and resolution is essential for optimal In‐cell NMR experiments.

Keywords: molecular crowding; posttranslational modifications; protein NMR; time‐resolved monitoring of cells; drug–receptor interactions; intrinsically disordered proteins; protein–protein interactions

Figure 1. Overview of in‐cell NMR sample preparation for observing isotopically enriched proteins in cells or cell extracts. See text for details.
Figure 2. Monitoring protein phosphorylation in cell extracts by NMR. (a) Chemical shift perturbations identify the phosphorylation site but are also sensed in neighbouring regions. (b) Phosphorylation of p53 protein in extracts by endogenous kinases of two cell types. (c) Phosphoserine signals in the unique domain of c‐Src in cytostatic factor (CSF) arrested X. laevis egg extracts showing that the PKA kinase inhibitor (H89) enhances the dephosphorylation of specific sites, by favouring the activation of phosphatases. Adapted by permission from Theillet et al. ©Nature Publishing Group.
Figure 3. Overview of fast NMR strategies to monitor posttranslational modifications in real time. The standard workflow for each ‘real time’ point implies repeated acquisition to increase signal‐to‐noise ratio and to acquire regularly spaced points from which spectra are obtained by Fourier transform. Fast methods include faster repetition rates and recording only a fraction of the points, which are enough to reconstruct the spectra using alternative processing methods. Minimising the time needed to record each individual spectrum increases the time resolution by which the biological events can be monitored but at the expense of spectral resolution. Mayzel et al. showed that time and frequency resolution can be optimised after the acquisition has been completed. See text for details.
Figure 4. Saturation transfer difference in whole cells. Binding of small molecules to receptors on the cell surface or other immobilised targets is monitored by the saturation of the NMR signals of small molecules when they are reversibly bound to macromolecules. The attenuation of the sharp signals from the free small molecules is detected by subtracting a reference spectrum. Attenuated signals are highlighted.
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Further Reading

Barbieri L, Luchinat E and Banci L (2014) Structural insights of proteins in sub‐cellular compartments: in‐mitochondria NMR. Biochimica et Biophysica Acta (BBA) – Molecular Cell Research 1843: 2492–2496.

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Luchinat E and Banci L (2016) A unique tool for cellular structural biology: in‐cell NMR. The Journal of Biological Chemistry 291: 3776–3784.

Luchinat E and Banci L (2017) In‐cell NMR: a topical review. IUCrJ 4: 108–118.

Pastore A and Temussi PA (2017) The Emperor's new clothes: myths and truths of in‐cell NMR. Archives of Biochemistry and Biophysics 628: 114–122.

Pielak GJ, Li C, Miklos AC, et al. (2009) Protein nuclear magnetic resonance under physiological conditions. Biochemistry 48: 226–234.

Reckel S, Löhr F and Dötsch V (2005) In‐cell NMR spectroscopy. Chembiochem 6: 1601–1606.

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Mateos, Borja, Maffei, Mariano, and Pons, Miquel(Apr 2018) In‐Cell NMR. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0027154]