Amide Hydrogen Deuterium Exchange in Isotopically Mixed Waters

TL;DR

This paper introduces a generalized model for hydrogen-deuterium exchange in protein amides within H2O/D2O mixtures, enabling more accurate interpretation of experimental data by accounting for back exchange and isotope effects.

Contribution

The authors develop a generalized Linderstrom-Lang framework that explicitly models forward and reverse exchange, improving analysis of HDX experiments in mixed isotopic solvents.

Findings

The GLL model accurately recovers protection factors in 50% D2O.

Ignoring back exchange underestimates protection factors.

Single mixed-solvent HDX experiments yield both conformational and local stability insights.

Abstract

Hydrogen-deuterium exchange (HDX) of protein backbone amides provides a powerful probe of conformational dynamics. However, when experiments are performed in H2O/D2O mixtures, quantitative interpretation is hindered by back exchange and isotope effects not captured by the classical Linderstrom-Lang (LL) model. We introduce a generalized Linderstrom-Lang (GLL) framework that explicitly accounts for forward and reverse exchange and for changes in protection upon isotopic substitution. Analytical solutions describe equilibrium enrichment (fractionation) and protection factors in mixtures, reducing to the LL model in pure D2O. Application to HDX/NMR of the molecular chaperone DNAJB1 in 50% D2O demonstrates that the GLL model recovers protection factors at 100% D2O. Ignoring back exchange (i.e., using the LL model) causes protection factors to be systematically underestimated. A particularly powerful feature of our approach is that a single HDX experiment in a mixture (e.g., 50% D2O) simultaneously provides protection factors that report on conformational dynamics and local stability, and fractionation factors that are sensitive to the local hydrogen-bonding environment.