Editing and diagonal peak suppression in three-dimensional HCCH protein NMR correlation experiments

A novel three-dimensional (3D) HCCH NMR experiment is introduced. It involves C-13-C-13 COSY or TOCSY coherence transfer plus two independent editing steps according to the number of protons attached to the individual carbons before and after the C-13-C-13 homonuclear mixing. This double editing leads to simplification of HCCH protein side chain spectra that otherwise are prone to spectral overlap. Another interesting feature is amino acid selectivity, i.e. that the presence of certain correlations in a doubly edited HCCH subspectrum gives a clue as to assignment to a particular subgroup of amino acids or segments thereof. Finally, the selection of two different multiplicities in the two editing steps leads to diagonal peak suppression in the H-1-H-1 (3D spectrum recorded with two H-1 and one C-13 dimension) or the C-13-C-13 (3D spectrum recorded with one H-1 and two C-13 dimensions) two-dimensional projection. The new experiment is demonstrated using a C-13,N-15-labeled protein sample, chymotrypsin inhibitor 2, at 500 MHz.