Kang C (2019), Applications of In-Cell NMR in Structural Biolo...

XB-ART-55588

Int J Mol Sci 2019 Jan 02;201:. doi: 10.3390/ijms20010139.

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Applications of In-Cell NMR in Structural Biology and Drug Discovery.

Kang C .

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In-cell nuclear magnetic resonance (NMR) is a method to provide the structural information of a target at an atomic level under physiological conditions and a full view of the conformational changes of a protein caused by ligand binding, post-translational modifications or protein⁻protein interactions in living cells. Previous in-cell NMR studies have focused on proteins that were overexpressed in bacterial cells and isotopically labeled proteins injected into oocytes of Xenopus laevis or delivered into human cells. Applications of in-cell NMR in probing protein modifications, conformational changes and ligand bindings have been carried out in mammalian cells by monitoring isotopically labeled proteins overexpressed in living cells. The available protocols and successful examples encourage wide applications of this technique in different fields such as drug discovery. Despite the challenges in this method, progress has been made in recent years. In this review, applications of in-cell NMR are summarized. The successful applications of this method in mammalian and bacterial cells make it feasible to play important roles in drug discovery, especially in the step of target engagement.

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Species referenced: Xenopus laevis
Genes referenced: fkbp1a

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	Figure 1. Sample preparation for in-cell NMR studies. The cells used for in-cell NMR studies can be prepared using the following strategies: Proteins (green) can be directly over-expressed in different cell lines using expression vectors. To make isotopically labeled proteins for in-cell NMR studies, the target gene can be cloned into suitable vectors followed with transfection/transformation into cells. Target protein can be isotopically labeled by growing cells in isotopically enriched (15N, 15N/13C) media. Cells with the overexpressed protein are then used for in-cell NMR experiments. Isotopically labeled proteins can also be prepared in vitro by overexpressing them in different cells or using cell-free expression systems. The labeled protein is then purified before being introduced to oocytes by microinjection. Blue box indicates the NMR tube. Labeled proteins can also be introduced into human cells using either cell-penetrating peptides (CPP), cell permeabilization by pore-forming toxins or electroporation as introduced previously [107]. This figure was modified from the figure of Luchinat and Banci [107].
	Figure 2. 3D NMR spectra collected in sf9 cells. (a) 3D HNCOCA (red), HNCA (black) and HNCO spectra of GB1 in sf9 cells. Blue box indicate the C’ signals. (b) Selected strip plot at 1HN−1H dimensions from the 3D a 15N-separated NOESY spectrum of GB1 expressed in sf9 cells. The Cα connectivity and sequential NOEs are indicated as blue and red lines, respectively. This figure was reprinted with permission from the reference [71]. Copyright (2013) American Chemical Society. For more experimental details, please refer to the original publication.
	Figure 3. Application of in-cell NMR in compound screening. Peptides that can disrupt FKBP12 and FRB interactions were screened using this approach. (a) 1H-15N-HSQC spectrum of FKBP12-FRB complex in which FKBP12 is 15N-labeled. (b) Peptide affects the spectrum of FKBP12-FRB complex. This figure was reprinted with permission from the reference [84]. Copyright (2009) American Chemical Society. For more experimental details, please refer to the original publication.

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