Motions of proteins are essential for the performance of their functions. Aliphatic protein side chains and their motions play critical roles in protein interactions: for recognition and binding of partner molecules at the surface or serving as an entropy reservoir within the hydrophobic core. Here, we present a new NMR method based on high-resolution relaxometry and high-field relaxation to determine quantitatively both motional amplitudes and time scales of methyl-bearing side chains in the picosecond-to-nanosecond range. We detect a wide variety of motions in isoleucine side chains in the protein ubiquitin. We unambiguously identify slow motions in the low nanosecond range, which, in conjunction with molecular dynamics computer simulations, could be assigned to transitions between rotamers. Our approach provides unmatched detailed insight into the motions of aliphatic side chains in proteins and provides a better understanding of the nature and functional role of protein side-chain motions.

Nuclear magnetic resonance (NMR) is a ubiquitous branch of spectroscopy that can explore matter at the scale of an atom. Significant improvements in sensitivity and resolution have been driven by a steady increase of static magnetic field strengths. However, some properties of nuclei may be more favourable at low magnetic fields. For example, transverse relaxation due to chemical shift anisotropy increases sharply at higher magnetic fields leading to line-broadening and inefficient coherence transfers. Here, we present a two-field NMR spectrometer that permits the application of rf-pulses and acquisition of NMR signals in two magnetic centres. Our prototype operates at 14.1 T and 0.33 T. The main features of this system are demonstrated by novel NMR experiments, in particular a proof-of-concept correlation between zero-quantum coherences at low magnetic field and single quantum coherences at high magnetic field, so that high resolution can be achieved in both dimensions, despite a ca. 10 ppm inhomogeneity of the low-field centre. Two-field NMR spectroscopy offers the possibility to circumvent the limits of high magnetic fields, while benefiting from their exceptional sensitivity and resolution. This approach opens new avenues for NMR above 1 GHz.