High-Pressure NMR Experiments for Detecting Protein Low-Lying Conformational States

High pressure is the method of choice for changing the relative population of a protein conformational sample. It is extremely useful for isolating and characterizing high-energy conformational states. Compared to all the perturbation methods, like pH or temperature, pressure perturbation's easy to apply and fully reversible.

It is also a local perturbation, primarily affecting regions with glass cavities or void volumes. To begin, introduce the nitrogen-15-labeled sample into the zirconia tube using a glass pipette, and ensure the sample seats at the bottom of the tube. To prevent the sample from mixing with the transmission liquid, overlay the sample with 200 microliters of mineral oil, and then fill the rest of the tube with transmission liquid.

Put a single-use O-ring on top of the zirconia tube and slide the tube into the base. Then connect the tube to the high-pressure tether line and tighten the base to the cell by hand. Then apply 14.7 newton meters torque to prevent leaks at lower pressure.

To check the integrity of the pressure cell assembly, pressurize the tube up to 300 bars outside the spectrometer using cell support and containment vessel. After 15 minutes, reset the pressure to one bar and check for leaks with a clean lint-free wipe. Insert the unpressurized tube into the spectrometer by carefully guiding the tether line.

Slide the tube into the spectrometer until the tube reaches sample sitting position. Lock, shim, match and tune the protium and nitrogen channels and save the optimized shims for future. Set up transverse relaxation optimized spectroscopy with hetero single-quantum coherent spectroscopy and proceed with recording a reference experiment at the atmospheric conditions.

Record a series of 2D experiments from one bar to 2.5 kilobars for every 500 bars. If the precise folding or unfolding rates are known, after each 500 bars'increment, let the sample equilibrate for 15 to 20 minutes. When the inflection point of the folding or unfolding transition reaches, record additional experiments to improve the precision of the fit.

This protocol was used to probe the pressure dependence of RRM2, the second RNA recognition motif heterogeneous nuclear ribonucleoprotein A1.Transverse relaxation optimized spectroscopy with hetero single-quantum coherence spectroscopy spectra was collected at one bar, 1.5 kilobars, two kilobars, and 2.5 kilobars. The RRM2 is almost completely unfolded within the 2.5 kilobar range. Individual pressure intensity profiles were fitted according to the equation shown here, to obtain the corresponding changes in the standard Gibbs free energy and volume associated with the unfolding reaction.

When mapped on the domain structure, the residues with the largest magnitude of volume change were found within the domain structural core, while those with the smallest magnitude of volume change were mainly located in the connecting loops between the beta strands and beta strand to the C terminal helix. To probe the degree of compressibility and conformational heterogeneity of the folded state ensemble, the protium chemical shifts were analyzed. Individual profiles of protium chemical shifts as a function of pressure were fitted using the equation shown, to extract the site-specific linear and nonlinear coefficients.

The residues with the largest nonlinear coefficients were mostly found in the structural core of the domain, while residues with the smallest nonlinear coefficients were mostly located within loops connecting the different structural motifs. It is important to set up the tube as a pressure set correctly, to prevent leaks. If a leak happens, the probe would probably have to be removed to clean up any trace of mineral oil.