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

Abstract

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.

Keywords: magnetization; methyl groups; nuclear magnetic resonance; proteins; residual dipolar couplings.