An optimized relaxation-based coherence transfer NMR experiment for the measurement of side-chain order in methyl-protonated, highly deuterated proteins.

Relaxation violated coherence transfer NMR spectroscopy has emerged as a powerful experimental tool for the quantitative measurement of amplitudes of motion of methyl containing side-chains. Typically, the experiments, performed on proteins that are highly deuterated and methyl-protonated, monitor the build-up of methyl (1)H double-quantum magnetization. Because all three protons in a methyl group are degenerate, such coherences can only result from differential relaxation of transverse magnetization components, which in turn reflect the extent and time-scale of motion of the methyl probe [Tugarinov, V., Sprangers, R.; Kay, L.E. J. Am. Chem. Soc. 2007, 129, 1743-1750]. We show here that a 50% gain in the sensitivity of the experiment can be achieved through selection of (1)H triple-quantum coherence, thereby significantly increasing the utility of the approach. A theoretical treatment rationalizes the sensitivity gain that is subsequently verified through experiment. The utility of the methodology is demonstrated on a number of proteins, including the 360 kDa α(7)α(7) "half-proteasome".