Exceeding the limit of dynamics studies on biomolecules using high spin-lock field strengths with a cryogenically cooled probehead.

Internal motions in the microsecond timescale have been proposed to play an active part in a protein's biological function. Nuclear magnetic resonance (NMR) relaxation dispersion is a robust method sensitive to this timescale with atomic resolution. However, due to technical limitations, the observation of motions faster than ∼40 μs for ¹⁵N nuclei was not possible. We show that with a cryogenically cooled NMR probehead, a high spin-lock field strength can be generated that is able to detect motions as fast as 25 μs. We apply this high spin-lock field strength in an NMR experiment used for characterizing dynamical processes. An on-resonance rotating-frame transverse relaxation experiment was implemented that allows for the detection of a 25 μs process from a dispersion curve, and transverse relaxation rates were compared at low and high spin-lock field strengths showing that at high field strengths contributions from chemical exchange with lifetimes up to 25 μs can be removed. Due to the increase in sensitivity towards fast motion, relaxation dispersion for a residue that undergoes smaller chemical shift variations due to dynamics was identified. This technique reduces the previously inaccessible window between the correlation time and the relaxation dispersion window that covers four orders of magnitude by a factor of 2.