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)CH(3) methyl groups in proteins, enabling the manipulation of the magnetization of a (13)CH(3) moiety as if it were an AX ((1)H-(13)C) spin-system. These experiments result in the same ‘simplification’ of a (13)CH(3) spin-system that would be obtained from the production of {(13)CHD(2)}-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)CHD(2)}-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)CH(3) (AX(3)) spin-systems are described, with an emphasis on the measurements of methyl (1)H-(13)C residual dipolar couplings in a {(13)CH(3)}-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.