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 ¹³CH₃ methyl groups in proteins, enabling the manipulation of the magnetization of a ¹³CH₃ moiety as if it were an AX (¹H-¹³C) spin-system. These experiments result in the same ‘simplification’ of a ¹³CH₃ spin-system that would be obtained from the production of {¹³CHD₂}-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 {¹³CHD₂}-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 ¹³CH₃ (AX₃) spin-systems are described, with an emphasis on the measurements of methyl ¹H-¹³C residual dipolar couplings in a {¹³CH₃}-methyl-labeled deletion mutant of the human chaperone DNAJB6b, where modulation of NMR signal intensities due to evolution of methyl ¹H-¹³C scalar and dipolar couplings follows a simple cosine function characteristic of an AX (¹H-¹³C) spin-system, significantly simplifying data analysis.