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Protein-observed 19F-NMR for fragment screening, affinity quantification and druggability assessment

Nature Protocols volume 11, pages 1414–1427 (2016)Cite this article

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Abstract

NMR spectroscopy can be used to quantify the binding affinity between proteins and low-complexity molecules, termed 'fragments'; this versatile screening approach allows researchers to assess the druggability of new protein targets. Protein-observed 19F-NMR (PrOF NMR) using 19F-labeled amino acids generates relatively simple spectra that are able to provide dynamic structural information toward understanding protein folding and function. Changes in these spectra upon the addition of fragment molecules can be observed and quantified. This protocol describes the sequence-selective labeling of three proteins (the first bromodomains of Brd4 and BrdT, and the KIX domain of the CREB-binding protein) using commercially available fluorinated aromatic amino acids and fluorinated precursors as example applications of the method developed by our research group. Fragment-screening approaches are discussed, as well as Kd determination, ligand-efficiency calculations and druggability assessment, i.e., the ability to target these proteins using small-molecule ligands. Experiment times on the order of a few minutes and the simplicity of the NMR spectra obtained make this approach well-suited to the investigation of small- to medium-sized proteins, as well as the screening of multiple proteins in the same experiment.

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Figure 1
Figure 2: PrOF NMR examples.
Figure 3: Characterization of fluorinated proteins expressed according to the present Protocol.
Figure 4: Using site-directed mutagenesis to assign PrOF resonances (see directions in Box 4).
Figure 5: Results from experiments involving small-molecule ligands for BrdT.
Figure 6: Deconvolution of fragment mixture.
Figure 7: Small-molecule titrations for Kd determination.

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Acknowledgements

This project was funded in part by the NSF-CAREER Award CHE-1352091 (to W.C.K.P., N.K.M. and L.M.L.H.), National Institutes of Health (NIH) Biotechnology training grant 5T32GM008347-23 (to A.K.U.) and NIH chemistry–biology interface training grant T32-GM08700 (to C.T.G.). The Pomerantz lab also thanks the Garber family and relatives for their generous support of this research. We also thank I. Ropson (Penn State University) for providing the DL39(DE3) cell line.

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Authors and Affiliations

  1. Department of Chemistry, University of Minnesota, Minneapolis, Minnesota, USA

    Clifford T Gee, Keith E Arntson, Andrew K Urick, Neeraj K Mishra, Laura M L Hawk & William C K Pomerantz

  2. Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota, USA

    Andrea J Wisniewski

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Contributions

W.C.K.P. oversaw the implementation and design of all experiments reported here. C.T.G. and K.E.A. carried out protein expression and characterization experiments with KIX; C.T.G. also carried out protein expression and characterization experiments with BrdT; N.K.M. and A.K.U. carried out protein expression and characterization experiments with Brd4; L.M.L.H. carried out fluorescence polarization experiments; and A.W. discovered fragment A. All authors assisted with writing and editing the manuscript.

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Correspondence to William C K Pomerantz.

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Integrated supplementary information

Supplementary Figure 1 Binding activity comparison of non-fluorinated and fluorinated proteins

Direct binding experiment of BODIPY-BI, a Brd4 bromodomain ligand to unlabeled Brd4 and 5FW-labeled Brd4 by fluorescence polarization.

Supplementary Figure 2 19F NMR spectral analysis of 5FW BrdT in the presence of increasing concentrations of Dinaciclib.

A) Left: Ribbon diagram of BrdT (PDB Code: 4FLP) with tryptophan side chains indicated as sticks shown in red. Right: Stacked PrOF NMR spectra with increasing concentration of dinaciclib. B) Absolute value of chemical shift perturbations for all 5FW BrdT tyrosine resonances at 400 μM Dinaciclib. C) Binding isotherm of both W44* and W50* perturbations for the titration with Dinaciclib. * denotes resonances that have not yet been assigned by site directed mutagenesis, but were inferred from ligand binding.

Supplementary Figure 3 19F NMR spectral analysis of 5FW BrdT in the presence of increasing concentrations of A.

A) Left: Ribbon diagram of BrdT (PDB Code: 4FLP) with tryptophan side chains indicated as sticks shown in red. Right: Stacked PrOF NMR spectra with increasing concentration of A. B) Absolute value of chemical shift perturbations for all 5FW BrdT tyrosine resonances at 2 mM A. C) Binding isotherm of both W44* and W50* perturbations for the titration with A. * denotes resonances that have not yet been assigned by site directed mutagenesis, but were inferred from ligand binding.

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Supplementary Figures 1–3, Supplementary Method, Supplementary Tables 1 and 2 (PDF 1604 kb)

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Gee, C., Arntson, K., Urick, A. et al. Protein-observed 19F-NMR for fragment screening, affinity quantification and druggability assessment. Nat Protoc 11, 1414–1427 (2016). https://doi.org/10.1038/nprot.2016.079

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