NMR Assignment of Methyl Groups in Immobilized Proteins Using Multiple-Bond 13C Homonuclear Transfers, Proton Detection, and Very Fast MAS

In nuclear magnetic resonance spectroscopy of proteins, methyl protons play a particular role as extremely sensitive reporters on dynamics, allosteric effects, and protein–protein interactions, accessible even in high-molecular-weight systems approaching 1 MDa. Themnotorious issue of their chemical shift assignment is addressed here by a joint use of solid-mstate 1 H-detected methods at very fast (nearly 100 kHz) magic-angle spinning, partialmdeuteration, and high-magnetic fields. The suitability of a series of RF schemes is evaluatedmfor the efficient coherence transfer across entire 13 C side chains of methyl-containing residues, which is key for establishing connection between methyl and backbone 1 H resonances. The performance of ten methods for recoupling of either isotropic 13 C–13 C scalar or anisotropic dipolar nteractions (five variants of TOBSY, FLOPSY, DIPSI, WALTZ,mRFDR, and DREAM) is evaluated experimentally at two state-of-the-art magic-angle spinning (55 and 94.5 kHz) and static magnetic field conditions (18.8 and 23.5 T). Model isotopically labeled compounds (alanine and Met-Leu-Phe tripeptide) and ILV-mmethyl and amide-selectively protonated, and otherwise deuterated chicken α-spectrin SH3 protein are used as convenient reference systems. Spin dynamics simulations in SIMPSON are performed to determine optimal parameters of these RF schemes, up to recently experimentally attained spinning frequencies (200 kHz) and B0 field strengths (28.2 T). The concept of linearization of 13 C side chain by ppropriate isotope labeling is revisited and showed to significantly increase sensitivity of methyl-to-backbone correlations. A resolution enhancement provided by 4D spectroscopy with non-uniform (sparse) sampling is demonstrated to remove ambiguities in simultaneous resonance assignment of methyl proton and carbon chemical shifts.