Amide Hydrogen Deuterium Exchange in Isotopically Mixed Waters

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.

Figures (4)

• Figure 1: Energy diagram for the GLL model \ref{['eq:gll']}. Exchange in the EX2 limit (high protection and exchange slower than opening/closing dynamics) is considered in two hypothetical mixtures at same pH and temperature and different compositions, 50% (magenta) and 90% (blue) D2O. Rate constants $k^\ddagger$ are related to the height of the corresponding barriers $\Delta G^\ddagger$ by Eyring equation: $k^\ddagger = (k_\mathrm{B}T/h) \mathrm{e}^{-\Delta G^\ddagger/RT}$. The solvent composition affects $k_\mathrm{forw}$, $k_\mathrm{back}$ and their ratio $K_\mathrm{back}$, determining the equilibrium between open states. A change in opening free energy upon isotopic substitution that is quantified by $\delta$, cfr Eq. \ref{['eq:delta-delta-G']}, affects equilibrium between deuterated states. The height of the barrier depends on $k_\mathrm{cl}'$ and $k_\mathrm{op}'$. In this illustration, it is assumed $k_\mathrm{cl}'=k_\mathrm{cl}$ and $k_\mathrm{op}'$ varying according to $\delta$.
• Figure 2: Experimental results for HDX/NMR of ^15N-DNAJB1 JD-GF-${\rm \alpha 5}$ F94L, at 25° C in 50% D2O and $\mathrm{pH_{read}} = 7.40$ and 100% D2O and $\mathrm{pH_{read} = 7.47}$. (A) Measured kinetics of residue L11, in 50% (magenta) and 100% (blue) D2O. The normalized intensity is the fraction of unexchanged amides, $1-D(t)$. (B) Protection factors ($\ln P$) estimated by measurements in 100% D2O. (C) Fractionation factors determined from equilibrium values in 50% D2O.
• Figure 3: Results obtained by the generalised Linderstrøm-Lang model for HDX/NMR of ^15N-DNAJB1 JD-GF-${\rm \alpha 5}$ F94L, performed in 50% D2O. (A) Difference in local stability (protection factors) upon isotopic substitution quantified by $\Delta\Delta G_\mathrm{op}$. (B) Inferred protection factors for undeuterated ($\ln P$) and deuterated ($\ln P'$) amides.
• Figure 4: HDX experimental data (dots) obtained from measurements in 50% D2O for residues (A) L11, and (B) Y6, and reproduced by the GLL model. Solid lines are generated from the full model, in which $\delta \neq 0$ indicates variation in local stability upon isotopic substitution and results in fractionation, as well as minor alteration to the kinetics. Dashed lines are obtained considering same $k_\mathrm{int,mix}$ and $P$, and $\delta = 0$. (C) Pair plot of protection factors extracted from data in 100% D2O versus protection factors estimated by LL (gray dots) and GLL (black dots) model from data in 50% D2O. The GLL model results present no systematic bias, while protection factors computed using the LL model are systematically underestimated.