Detecting Protein‐Ligand Interactions with Nitroxide Based Paramagnetic Cosolutes

Abstract

NMR spectroscopy techniques can provide important information about protein‐ligand interactions. Here we tested an NMR approach which relies on the measurement of paramagnetic relaxation enhancements (PREs) arising from analogous cationic, anionic or neutral soluble nitroxide molecules, which distribute around the protein‐ligand complex depending on near‐surface electrostatic potentials. We applied this approach to two protein‐ligand systems, interleukin‐8 interacting with highly charged glycosaminoglycans and the SH2 domain of Grb2 interacting with less charged phospho‐tyrosine tripeptides. The electrostatic potential around interleukin‐8 and its changes upon binding of glycosaminoglycans could be derived from the PRE data and confirmed by theoretical predictions from Poisson‐Boltzmann calculations. The ligand influence on the PREs and NMR‐derived electrostatic potentials of Grb2 SH2 was localized to a narrow protein region which allowed the localization of the peptide binding pocket. Our analysis suggests that experiments with nitroxide cosolutes can be useful for investigating protein‐ligand electrostatic interactions and mapping ligand binding sites.

Gadolinium-Based NMR Spin Relaxation Measurements of Near-Surface Electrostatic Potentials of Biomolecules

“…Because the ϕ ENS method does not require any structural information, its applicability extends to conformationally flexible systems, including IDRs of proteins . For example, the ϕ ENS method has been applied to map per-residue surface electrostatic potentials of the positively charged 103 residue carboxyl-terminus of the 702 residue RNA-binding protein CAPRIN1 along its phase-separation trajectory. , This NMR method has also been used to investigate the electrostatic properties of the unfolded drk SH3 domain and the disordered Pmel17 repeat domain, as well as electrostatic interactions between a number of different proteins and their conformationally flexible ligands …”

“… 22 , 23 This NMR method has also been used to investigate the electrostatic properties of the unfolded drk SH3 domain 24 and the disordered Pmel17 repeat domain, 25 as well as electrostatic interactions between a number of different proteins and their conformationally flexible ligands. 26 …”