Spectral editing of intra- and inter-chain methyl-methyl NOEs in protein complexes

Abstract

Specific isotopic labeling of methyl groups in a perdeuterated protein background enables the detection of long range NOEs in proteins or high molecular weight complexes. We introduce here an approach, combining an optimized isotopic labeling scheme with a specifically tailored NMR pulse sequence, to distinguish between intramolecular and intermolecular NOE connectivities. In hetero-oligomeric complexes, this strategy enables sign encoding of intra-subunit and inter-subunit NOEs. For homo-oligomeric assemblies, our strategy allows the specific detection of intra-chain NOEs in high resolution 3D NOESY spectra. The general principles, possibilities and limitations of this approach are presented. Applications of this approach for the detection of intermolecular NOEs in a hetero-hexamer, and the assignment of methyl ¹H and ¹³C resonances in a homo-tetrameric protein complex are shown.

Keywords: Assignment; Isotopic labeling; Large protein; Long range NOE; Methyl groups; NMR; Protein complex; Structure determination.