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

Summary

We provide a detailed description of the steps required to assemble a high-pressure cell, set up and record high-pressure NMR experiments, and finally analyze both peak intensity and chemical shift changes under pressure. These experiments can provide valuable insights into the folding pathways and structural stability of proteins.

Abstract

High-pressure is a well-known perturbation method that can be used to destabilize globular proteins and dissociate protein complexes in a reversible manner. Hydrostatic pressure drives thermodynamical equilibria toward the state(s) with the lower molar volume. Increasing pressure offers, therefore, the opportunities to finely tune the stability of globular proteins and the oligomerization equilibria of protein complexes. High-pressure NMR experiments allow a detailed characterization of the factors governing the stability of globular proteins, their folding mechanisms, and oligomerization mechanisms by combining the fine stability tuning ability of pressure perturbation and the site resolution offered by solution NMR spectroscopy. Here we present a protocol to probe the local folding stability of a protein via a set of 2D 1H-15N experiments recorded from 1 bar to 2.5 kbar. The steps required for the acquisition and analysis of such experiments are illustrated with data acquired on the RRM2 domain of hnRNPA1.

Introduction

It has long been recognized that higher-energy, sparsely populated conformational states of proteins and protein complexes play a key role in many biological pathways^1,2,3. Thanks to experiments based on Carr-Purcell-Meiboom-Gill (CPMG)⁴, Chemical Exchange Saturation Transfer (CEST)⁵, and dark-state exchange saturation transfer (DEST)⁶ pulse sequences (among others), solution NMR spectroscopy has emerged as a method of choice for characterizing transient conformational states⁷. Alo....

Protocol

NOTE: The protocol described here requires (i) a high-pressure pump and cell with a 2.5 kbar rated aluminum-toughened zirconia tube¹⁸, (ii) the software SPARKY¹⁹ for analysis of the NMR spectra, and (iii) a curve fitting software.

1. Sample preparation, assembly of the high-pressure cell, and setting up the experiments.

1. Choice of buffer: Use equal mixture of anionic and cationic buffers, such as phosphate and Tris^20,21.
   NOTE: The pKa of anionic buffers such as phosphate and MES is associated with a substantial reacti....

Results

The protocol described here was used to probe the pressure dependence of RRM2, the second RNA recognition motif of hnRNPA1 (residues 95-106), which is almost completely unfolded within the 2.5 kbar range (>90%). ¹H-¹⁵N spectra were collected at 1 bar, 500 bar, 750 bar, 1 kbar, 1.5 kbar, 2 kbar, and 2.5 kbar (Figure 2). Since none of the native crosspeaks were visible above the noise level at 2.5 kbar, all corresponding residues were attributed an intensity valu.......

Discussion

This study details a protocol implemented to probe protein structural and thermodynamics responses to pressure perturbation. The high-pressure experiments recorded here on RRM2 demonstrate that large variations in ΔV_U values, indicative of non-fully cooperative unfolding, can be found in a relatively small single domain protein. A similar picture emerges from the analysis of ¹H chemical shift changes under pressure. It should be noted Kalbitzer and coworkers have demonstrated that a more i.......

Materials

Name	Company	Catalog Number	Comments
Bruker Nmr Cell 2.5 Kbar	Daedalus Innovations LLC	NMRCELL-B
Sparky3	University of California San Francisco, CA	N/A
Xtreme-60 Syringe pump	Daedalus Innovations LLC	XTREME-60