Abstract
Among other perturbations, high hydrostatic pressure has proven to be a mild yet efficient way to unfold proteins. Combining pressure perturbation with NMR spectroscopy allows for a residue-per-residue description of folding reactions. Accessing the full power of NMR spectroscopy under pressure involves the investigation of conformational sampling using orientational restraints such as residual dipolar couplings (RDCs) under conditions of partial alignment. The aim of this study was to identify and characterize stable and pressure resistant alignment media for measurement of RDCs at high pressure. Four alignment media were tested. A C12E5/n-hexanol alcohol mixture remains stable from 1 to 2,500 bar, whereas Pf1 phage and DNA nanotubes undergo a reversible transition between 300 and 900 bar. Phospholipid bicelles are stable only until 300 bar at ambient temperature. Hence, RDCs can be measured at high pressure, and their interpretation will provide atomic details of the structural and dynamic perturbations on unfolded or partially folded states of proteins under pressure.
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Acknowledgments
The authors are indebted to Drs. P. Bernadó for valuable discussion, F. Allemand for sample preparation and G. Bellot for providing DNA nanotubes. We gratefully acknowledge support from the Agence National de la Recherche, Grant PiriBio 09-455024. High pressure NMR work was supported by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01 and the Structural Biology RIO/IbiSA platform (Centre de Biochimie Structurale).
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Sibille, N., Dellarole, M., Royer, C. et al. Measuring residual dipolar couplings at high hydrostatic pressure: robustness of alignment media to high pressure. J Biomol NMR 58, 9–16 (2014). https://doi.org/10.1007/s10858-013-9798-7
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DOI: https://doi.org/10.1007/s10858-013-9798-7