Accurate Measurements of Solvent Exchange in Asparagine and Glutamine Side-Chain NH 2 Groups of Proteins by z -Exchange NMR Spectroscopy

NMR methodology is developed for accurate measurements of solvent exchange rates in asparagine and glutamine side-chain NH2 groups of proteins by z-exchange spectroscopy. A rigorous model of solvent exchange in NH2 groups of proteins dissolved in a 50%/50% (v/v) mixture of H2O/D2O solvents, that incorporates the independently measured rates of hindered rotation around the carboxamide C'-N bond, allowed determining solvent exchange rates for side-chain amides of the model proteins ubiquitin and GB1, as well as a subset of NH2 groups of a buried cavity mutant of T4 lysozyme. In agreement with earlier findings, the amide protons at E (anti) positions usually exchange faster with the solvent than their Z (syn) counterparts. The temperature dependence of the rates of hindered rotation and solvent exchange in ubiquitin showed that the two processes are enthalpically driven and characterized by very similar activation enthalpies of ∼70 kJ/mol.