Relaxation dispersion techniques are powerful tools to quantitatively characterize the chemical (or conformational) exchange across biologically relevant timescales [Palmer et al, Meth. Enzymol. 2001 & 2019]. Recently, a new type of data analysis, geometric approximation methodology [Chao & Byrd, JACS 2016], has been developed to decipher the complex experimental data associated with the adiabatic relaxation dispersion experiment [Mangia et al. JACS 2010], thus providing the assessment of a very broad range of timescales. The advantages of geometric approximation can also be applied to conventional CPMG experiments, including the use of different exchange models and related problems [Chao & Byrd, JMR 2017, Emerg. Topics Life Sci., 2018].
The original adiabatic relaxation dispersion experiment focused on protein backbone (15NH) dynamics. We have recently extended these methods to methyl groups in the new methyl-geoHARD experiment, combining geometric approximation, adiabatic relaxation dispersion techniques, and methyl TROSY effects [Tugarinov et al. JACS 2003]. Methyl-geoHARD can explore broad timescale conformational dynamics (ranging from 150 sec-1 to 100,000 sec-1) in the hydrophobic cores of large protein complexes.
We illustrate the detection and quantification of a broad distribution of collective motions and local motions of methyl groups within a moderately large enzyme (tauc = 24 ns). The method is quantitatively validated by comparison with the conventional SQ-CPMG [Lundstrom et al., JBNMR 2007] and other experimental data, for sites where the dynamics are within the timescale of both experiments. The technique is developed and optimized to address the large off-resonance effects at ultra-high magnetic fields (> 1GHz) and for large and complex biological systems (up to tauc = 50 ns). Overall, the potentials of geometric approximation methodology enable the analysis of complex relaxation phenomena and simplify the experiments to gain or retain sensitivity in challenging, large molecular weight proteins.