Issue 11, 2019

Selective high-resolution DNP-enhanced NMR of biomolecular binding sites

Abstract

Locating binding sites in biomolecular assemblies and solving their structures are of the utmost importance to unravel functional aspects of the system and provide experimental data that can be used for structure-based drug design. This often still remains a challenge, both in terms of selectivity and sensitivity for X-ray crystallography, cryo-electron microscopy and NMR. In this work, we introduce a novel method called Selective Dynamic Nuclear Polarization (Sel-DNP) that allows selective highlighting and identification of residues present in the binding site. This powerful site-directed approach relies on the use of localized paramagnetic relaxation enhancement induced by a ligand-functionalized paramagnetic construct combined with difference spectroscopy to recover high-resolution and high-sensitivity information from binding sites. The identification of residues involved in the binding is performed using spectral fingerprints obtained from a set of high-resolution multidimensional spectra with varying selectivities. The methodology is demonstrated on the galactophilic lectin LecA, for which we report well-resolved DNP-enhanced spectra with linewidths between 0.5 and 1 ppm, which enable the de novo assignment of the binding interface residues, without using previous knowledge of the binding site location. Since this approach produces clean and resolved difference spectra containing a limited number of residues, resonance assignment can be performed without any limitation with respect to the size of the biomolecular system and only requires the production of one protein sample (e.g.13C,15N-labeled protein).

Graphical abstract: Selective high-resolution DNP-enhanced NMR of biomolecular binding sites

Supplementary files

Article information

Article type
Edge Article
Submitted
20 Dec 2018
Accepted
01 Feb 2019
First published
01 Feb 2019
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY-NC license

Chem. Sci., 2019,10, 3366-3374

Selective high-resolution DNP-enhanced NMR of biomolecular binding sites

I. Marin-Montesinos, D. Goyard, E. Gillon, O. Renaudet, A. Imberty, S. Hediger and G. De Paëpe, Chem. Sci., 2019, 10, 3366 DOI: 10.1039/C8SC05696J

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