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Automated projection spectroscopy (APSY) for efficient structural characterization of protein-ligand interactions

Gossert, Alvar and Hiller, Sebastian and Fernandez, Cesar (2010) Automated projection spectroscopy (APSY) for efficient structural characterization of protein-ligand interactions. Journal of the American Chemical Society. ISSN 0002-7863

Abstract

The detection and structural characterization of protein–ligand interactions is central to functional biology research as well as to drug discovery. In high-throughput settings, nuclear magnetic resonance (NMR) is widely used to detect compound binding events, but it has not found widespread application to yield structural information of protein–ligand binding interfaces, because such experiments are limited by the time-intensive human work required for sequence-specific resonance assignments. Here we present a robust and highly automated procedure for obtaining the resonance assignments necessary for efficient mapping of protein–ligand interactions in a routine setup. The procedure relies on a combination of three automated projection spectroscopy (APSY) experiments with an overall molecular size limit of beyond 25 kDa. This set of APSY experiments is recorded with perdeuterated protein samples in a total of 5 days of instrument time. Low signal-to-noise picking of the spectra with subsequent filtering of the reconstructed peak list is employed for optimal sensitivity. Since the recorded chemical shifts include the Calpha and Cbeta nuclei, these data allow a reliable and robust assignment of the protein backbone by linear algorithms supported by minimal human interaction. Structurally relevant side chains are included in these assignments, providing the basis for efficient characterization of protein–ligand interactions at atomic resolution. We show the successful application of this procedure to two human proteins with sizes of 15 and 22 kDa, and discuss the general applicability to studies of protein–protein and protein–RNA/DNA complexes.

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Date Deposited: 13 Oct 2015 13:17
Last Modified: 13 Oct 2015 13:17
URI: https://oak.novartis.com/id/eprint/1085

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