Arginine Side‐Chain Hydrogen Exchange: Quantifying Arginine Side‐Chain Interactions in Solution

The rate with which labile backbone hydrogen atoms in proteins exchange with the solvent has long been used to probe protein interactions in aqueous solutions. Arginine, an essential amino acid found in many interaction interfaces, is capable of an impressive range of interactions via its guanidinium group. The hydrogen exchange rate of the guanidinium hydrogens therefore becomes an important measure to quantify side‐chain interactions. Herein we present an NMR method to quantify the hydrogen exchange rates of arginine side‐chain (1)H(ϵ) protons and thus present a method to gauge the strength of arginine side‐chain interactions. The method employs (13)C‐detection and the one‐bond deuterium isotope shift observed for (15)N(ϵ) to generate two exchanging species in (1)H(2)O/(2)H(2)O mixtures. An application to the protein T4 Lysozyme is shown, where protection factors calculated from the obtained exchange rates correlate well with the interactions observed in the crystal structure. The methodology presented provides an important step towards characterising interactions of arginine side‐chains in enzymes, in phase separation, and in protein interaction interfaces in general.