15N,1H]/[13C,1H]-TROSY for simultaneous detection of backbone 15N-1H, aromatic 13C-1H and side-chain 15N-1H2 correlations in large proteins.

Abstract

This paper describes a [15N,1H]/[13C,1H]-TROSY experiment for the simultaneous acquisition of the heteronuclear chemical shift correlations of backbone amide 15N-1H groups, side chain 15N-1H2 groups and aromatic 13C-1H groups in otherwise highly deuterated proteins. The 15N-1H and 13C-1H correlations are extracted from two subspectra of the same data set, thus preventing possible spectral overlap of aromatic and amide protons in the 1H dimension. The side-chain 15N-1H2 groups, which are suppressed in conventional [15N,1H)-TROSY, are observed with high sensitivity in the 15N-1H subspectrum. [15N,1H]/[13C,1H]-TROSY was used as the heteronuclear correlation block in a 3D [1H,1H]-NOESY-[15N,1H]/[13C,1H]-TROSY experiment with the membrane protein OmpA reconstituted in detergent micelles of molecular weight 80000 Da, which enabled the detection of numerous NOEs between backbone amide protons and both aromatic protons and side chain 15N-1H2 groups.