Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for characterizing biomolecules such as proteins and nucleic acids at atomic resolution. Increased magnetic field strengths drive progress in biomolecular NMR applications, leading to improved performance, e.g., higher resolution. A new class of NMR spectrometers with a 28.2 T magnetic field (1.2 GHz 1H frequency) has been commercially available since the end of 2019. The availability of ultra-high-field NMR instrumentation makes it possible to investigate more complex systems using NMR. This is especially true for highly flexible intrinsically disordered proteins (IDPs) and highly flexible regions (IDRs) of complex multidomain proteins. Indeed, the investigation of these proteins is frequently hampered by the crowding of NMR spectra. The advantages, however, are accompanied by challenges that the user must overcome when conducting experiments at such a high field (e.g., large spectral widths, radio frequency bandwidth, performance of decoupling schemes). This protocol presents strategies and tricks for optimising high-field NMR experiments for IDPs/IDRs based on the analysis of the relaxation properties of the investigated protein. The protocol, tested on three IDPs of different molecular weight and structural complexity, focuses on 13C-detected NMR at 1.2 GHz. A set of experiments, including some multiple receiver experiments, and tips to implement versions tailored for IDPs/IDRs are described. However, the general approach and most considerations can also be applied to experiments that acquire 1H or 15N nuclei and to experiments performed at lower field strengths.
Key points: 28.2 T nuclear magnetic resonance spectrometers are now available and, thanks to their improved resolution, are especially useful for analyzing proteins that have flexible regions.At such high magnetic fields, there are important challenges relating to the concomitant increase in spectral width. Key points explored in this protocol include the relaxation properties of proteins, choice of pulses for excitation and decoupling and setup of two-dimensional and multiple receiver experiments.
The recently available 28.2 Tesla nuclear magnetic resonance spectrometers (1.2 GHz 1H frequency) can be used to characterize highly flexible proteins and protein regions. This protocol explains how to setup 13C nuclear magnetic resonance experiments to achieve optimal results.