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Hydration of denatured and molten globule proteins

Nature Structural Biology volume 6, pages 253–260 (1999)Cite this article

Abstract

The hydration of nonnative states is central to protein folding and stability but has been probed mainly by indirect methods. Here we use water 17O relaxation dispersion to monitor directly the internal and external hydration of α-lactalbumin, lysozyme, ribonuclease A, apomyoglobin and carbonic anhydrase in native and nonnative states. The results show that nonnative proteins are more structured and less solvent exposed than commonly believed. Molten globule proteins preserve most of the native internal hydration sites and have native-like surface hydration. Proteins denatured by guanidinium chloride are not fully solvent exposed but contain strongly perturbed occluded water. These findings shed new light on hydrophobic stabilization of proteins.

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Figure 1: Crystal structures of human α-lactalbumin (PDB file 1HML), lysozyme (2LZT) and ribonuclease A (7RSA) showing potentially long-lived internal water molecules, disulfide bonds (yellow), and the Ca2+ ion (green) in α-lactalbumin.
Figure 2: Frequency dependence of water 17O excess longitudinal relaxation rate from solutions of α-lactalbumin, lysozyme and ribonuclease A in the native (○), GdmCl-denatured (♦) and GdmCl-denatured-reduced (X) states.
Figure 3: Variation with GdmCl concentration of the hydration parameters NβSβ2 (left) and Nαρα (right) derived from 17O MRD profiles from α-lactalbumin solutions.
Figure 4: Frequency dependence of water 17O excess longitudinal relaxation rate from solutions of α-lactalbumin, apomyoglobin and carbonic anhydrase II in the native (○) and molten globule (●) states, and from solutions of acid-denatured apoMb (X) and GdmCl-denatured HCAII (X).
Figure 5: Circular dichroism spectra from aqueous solutions of the proteins studied in the MG state by MRD.
Figure 6: 1H NMR spectra from aqueous solutions of the proteins studied in the MG state by MRD: a, BαLA (N), b, BαLA (MG), c, apoMb (N), d, apoMb (MG), e, apoMb (D), f, HCAII (N), and g, HCAII (MG).

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Acknowledgements

We thank E. Thulin for help with protein purification. This work was supported by the Swedish Natural Science Research Council (NFR) and the Royal Swedish Academy of Sciences (KVA).

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Authors and Affiliations

  1. Department of Chemistry, Condensed Matter Magnetic Resonance Group, Lund University, Lund, S-22100, Sweden

    Vladimir P. Denisov & Bertil Halle

  2. Department of Biochemistry, Umeå University, Umeå, S-90187, Sweden

    Bengt-Harald Jonsson

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  1. Vladimir P. Denisov
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Correspondence to Bertil Halle.

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Denisov, V., Jonsson, BH. & Halle, B. Hydration of denatured and molten globule proteins. Nat Struct Mol Biol 6, 253–260 (1999). https://doi.org/10.1038/6692

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