Optimized NMR Experiments for the Isolation of I=1/2 Manifold Transitions in Methyl Groups of Proteins.

Optimized NMR experiments are developed for isolating magnetization belonging to the I=1/2 manifolds of 13 CH3 methyl groups in proteins, enabling the manipulation of the magnetization of a 13 CH3 moiety as if it were an AX (1 H-13 C) spin-system. These experiments result in the same 'simplification' of a 13 CH3 spin-system that would be obtained from the production of {13 CHD2 }-methyl-labeled protein samples. The sensitivity of I=1/2 manifold-selection experiments is a factor of approximately 2 less than that of the corresponding experiments acquired on {13 CHD2 }-labeled methyl groups. The methodology described here is primarily intended for small-to-medium sized proteins, where the losses in sensitivity associated with the isolation of I=1/2 manifold transitions can be tolerated. Several NMR applications that benefit from simplification of the 13 CH3 (AX3 ) spin-systems are described, with an emphasis on the measurements of methyl 1 H-13 C residual dipolar couplings in a {13 CH3 }-methyl-labeled deletion mutant of the human chaperone DNAJB6b, where modulation of NMR signal intensities due to evolution of methyl 1 H-13 C scalar and dipolar couplings follows a simple cosine function characteristic of an AX (1 H-13 C) spin-system, significantly simplifying data analysis.