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. 2018 Sep;27(9):1710-1716.
doi: 10.1002/pro.3477.

Enthalpic stabilization of an SH3 domain by D2 O

Samantha S Stadmiller  1 , Gary J Pielak  1   2   3   4
Affiliations

Enthalpic stabilization of an SH3 domain by D2 O

Samantha S Stadmiller et al. Protein Sci. 2018 Sep.

Abstract

The stability of a protein is vital for its biological function, and proper folding is partially driven by intermolecular interactions between protein and water. In many studies, H2 O is replaced by D2 O because H2 O interferes with the protein signal. Even this small perturbation, however, affects protein stability. Studies in isotopic waters also might provide insight into the role of solvation and hydrogen bonding in protein folding. Here, we report a complete thermodynamic analysis of the reversible, two-state, thermal unfolding of the metastable, 7-kDa N-terminal src-homology 3 domain of the Drosophila signal transduction protein drk in H2 O and D2 O using one-dimensional 19 F NMR spectroscopy. The stabilizing effect of D2 O compared with H2 O is enthalpic and has a small to insignificant effect on the temperature of maximum stability, the entropy, and the heat capacity of unfolding. We also provide a concise summary of the literature about the effects of D2 O on protein stability and integrate our results into this body of data.

Keywords: NMR spectroscopy; SH3 domain; deuterium oxide; protein folding; protein stability; solvent isotope effect; thermodynamics.

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Figures

Figure 1
Figure 1
Two‐state, temperature‐dependent, reversible SH3 folding in H2O and D2O monitored with 19F NMR. (A) One‐dimensional spectra in H2O at nine temperatures normalized to the intensity of the folded state peak at 5°C. Subsequent spectra are offset by −0.2 ppm to aid visualization. The downfield resonance is the folded state (F), and the upfield resonance is the unfolded ensemble (U). (B) Spectra at 25°C before (black) and after (gray) an experiment where the temperature ranged from 5°C to 45°C over approximately 180 min indicating the reversibility of denaturation. (C) Spectra in H2O (black) and D2O (red) at 45°C. The D2O spectrum is normalized to the chemical shift and intensity of the folded state resonance in H2O.
Figure 2
Figure 2
Temperature dependence of SH3 stability in H2O and D2O. Error bars represent standard errors of the mean from three independent trials.
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