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Molecules 2018, 23(8), 1937; https://doi.org/10.3390/molecules23081937

Probing Protein-Protein Interactions Using Asymmetric Labeling and Carbonyl-Carbon Selective Heteronuclear NMR Spectroscopy

1
Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA
2
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
3
National Magnetic Resonance Facility at Madison, Madison, WI 53706, USA
4
Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA
*
Author to whom correspondence should be addressed.
Academic Editor: Brian F. Volkman
Received: 10 July 2018 / Revised: 23 July 2018 / Accepted: 25 July 2018 / Published: 3 August 2018
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Abstract

Protein-protein interactions (PPIs) regulate a plethora of cellular processes and NMR spectroscopy has been a leading technique for characterizing them at the atomic resolution. Technically, however, PPIs characterization has been challenging due to multiple samples required to characterize the hot spots at the protein interface. In this paper, we review our recently developed methods that greatly simplify PPI studies, which minimize the number of samples required to fully characterize residues involved in the protein-protein binding interface. This original strategy combines asymmetric labeling of two binding partners and the carbonyl-carbon label selective (CCLS) pulse sequence element implemented into the heteronuclear single quantum correlation (1H-15N HSQC) spectra. The CCLS scheme removes signals of the J-coupled 15N–13C resonances and records simultaneously two individual amide fingerprints for each binding partner. We show the application to the measurements of chemical shift correlations, residual dipolar couplings (RDCs), and paramagnetic relaxation enhancements (PRE). These experiments open an avenue for further modifications of existing experiments facilitating the NMR analysis of PPIs. View Full-Text
Keywords: protein-protein interactions (PPI); nuclear magnetic resonance (NMR); Carbonyl Carbon Label Selective (CCLS); dual carbon label selective (DCLS); residual dipolar coupling (RDC); paramagnetic relaxation enhancement (PRE) protein-protein interactions (PPI); nuclear magnetic resonance (NMR); Carbonyl Carbon Label Selective (CCLS); dual carbon label selective (DCLS); residual dipolar coupling (RDC); paramagnetic relaxation enhancement (PRE)
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).
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Larsen, E.K.; Olivieri, C.; Walker, C.; V.S., M.; Gao, J.; Bernlohr, D.A.; Tonelli, M.; Markley, J.L.; Veglia, G. Probing Protein-Protein Interactions Using Asymmetric Labeling and Carbonyl-Carbon Selective Heteronuclear NMR Spectroscopy. Molecules 2018, 23, 1937.

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