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

Erik K Larsen; Cristina Olivieri; Caitlin Walker; Manu V S; Jiali Gao; David A Bernlohr; Marco Tonelli; John L Markley; Gianluigi Veglia

doi:10.3390/molecules23081937

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

Molecules. 2018 Aug 3;23(8):1937. doi: 10.3390/molecules23081937.

Authors

Erik K Larsen¹, Cristina Olivieri², Caitlin Walker³, Manu V S⁴, Jiali Gao⁵, David A Bernlohr⁶, Marco Tonelli⁷, John L Markley⁸, Gianluigi Veglia^{9

10}

Affiliations

¹ Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA. larse585@umn.edu.
² Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA. colivier@umn.edu.
³ Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA. walk0512@umn.edu.
⁴ Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA. mvelipar@umn.edu.
⁵ Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA. jgao@umn.edu.
⁶ Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA. bernl001@umn.edu.
⁷ National Magnetic Resonance Facility at Madison, Madison, WI 53706, USA. tonelli@nmrfam.wisc.edu.
⁸ Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA. markley@biochem.wisc.edu.
⁹ Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA. vegli001@umn.edu.
¹⁰ Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA. vegli001@umn.edu.

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.

Keywords: Carbonyl Carbon Label Selective (CCLS); dual carbon label selective (DCLS); nuclear magnetic resonance (NMR); paramagnetic relaxation enhancement (PRE); protein-protein interactions (PPI); residual dipolar coupling (RDC).

Publication types

Review

MeSH terms

Carbon Isotopes
Isotope Labeling / methods*
Nitrogen Isotopes
Nuclear Magnetic Resonance, Biomolecular*
Protein Binding
Protein Conformation
Proteins / chemistry*

Substances

Carbon Isotopes
Nitrogen Isotopes
Proteins

Abstract

Publication types

MeSH terms

Substances

Grants and funding