Measuring residual dipolar couplings at high hydrostatic pressure: robustness of alignment media to high pressure

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.

Pressure‐induced structural transition of mature HIV‐1 protease from a combined NMR/MD simulation approach

“…S2). Thus squalamine is an additional pressure‐stable alignment medium . RDC measurements were carried out both 1 bar and at a pressure close to the midpoint of denaturation, where ∼55% of the folded state is retained based on the relative intensities of the 1 H‐ 15 N cross peaks (Supporting Information Fig.…”

Combining High-Pressure Perturbation with NMR Spectroscopy for a Structural and Dynamical Characterization of Protein Folding Pathways

“…Compared with Yamada's cell, this set-up provides a similar spectral quality but about a 10-fold increase in sensitivity and is incomparably easier to handle, essentially due to a wider inner diameter facilitating its filling. Thus, these ceramic tubes can be easily filled with complex viscous samples, such as those used for RDC measurement [41][42][43]. Pressure is generally transmitted by mineral oil, so that no physical separation is needed between the aqueous buffer containing the protein and the transmitting fluid.…”

Combining High-Pressure Perturbation with NMR Spectroscopy for a Structural and Dynamical Characterization of Protein Folding Pathways

“…Compared to Yamada's cell, this set-up provides a similar spectral quality, but about ten-fold increase in sensitivity and is incomparably more easy to handle, essentially due to a wider inner diameter facilitating its filling. Thus, these ceramic tubes can be easily filled with complex viscous samples, such as those used for RDC measurement [41][42][43]. Pressure is generally transmitted by mineral oil, so that no physical separation is needed between the aqueous buffer containing the protein and the transmitting fluid.…”