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.
Highlights
Biological processes rely primarily on protein-protein interactions (PPIs) to mediate a cellular function [1]
Several new nuclear magnetic resonance (NMR) experiments based on simultaneous, interleaved detection of up to three NMR active species with distinct isotopic labeling have provided the opportunity to map the effect of Protein-protein interactions (PPIs) on individual components within a macromolecular complex
The spin-echo filtered experiment introduced by Bax is the building block for the Carbonyl Carbon Label Selective (CCLS) 1 H-15 N HSQC pulse sequence [32], which requires specific isotopic labeling to simultaneously map the chemical shift perturbations from two binding partners
Summary
Biological processes rely primarily on protein-protein interactions (PPIs) to mediate a cellular function [1]. The mapping of PPIs using several observables such as chemical shift perturbation (CSP), residual dipolar couplings (RDC), intra-molecular and inter-molecular as well as solvent paramagnetic relaxation enhancement (PRE) [10,11,12], cross-saturation (CS), and nuclear Overhauser effects (NOEs) has been well-established [5]. These methods, fall short when studying large complexes due to the inherent attenuation of transverse relaxation times (T2 ), which results in a reduction of both signal intensity and resolution. While there are outstanding reviews on protein-protein interactions [18,19,20,21,22,23,24], we focus on our recently developed method that exploits the spin-echo filtering-based experiments with strategic protein labeling schemes to characterize protein-protein complexes
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