Accurate Measurements of Solvent Exchange in Asparagine and Glutamine Side-Chain NH ₂ Groups of Proteins by <i>z</i> -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 <i>z</i>-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 <i>E</i> (<i>anti</i>) positions usually exchange faster with the solvent than their <i>Z</i> (<i>syn</i>) 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.