HIGH-RESOLUTION 3D HNCOCA EXPERIMENT APPLIED TO A 28-KDA PARAMAGNETICPROTEIN

A new triple-resonance 3D HNCOCA pulse scheme is presented, designed t o identify the backbone nuclei (H-N, N, CO, C-alpha) of doubly labelle d proteins. The two carbon frequencies are labelled along the same ind irect dimension and the corresponding dwell times can be independently scaled in order to account for the relaxation properties and chemical shift ranges of the CO and C-alpha. If one takes advantage of the sym metry properties of the spectra in the course of the peak picking, thi s 3D scheme has the same sensitivity as the 4D experiment, but with an improved resolution The sequence is illustrated on a 0.5 mM sample of Rhodobacter capsulatus cytochrome c', a homodimeric paramagnetic prot ein of 2 x 14 kDa. A resonance assignment strategy, based on a low-con centration C-13/N-15-labelled sample and a more concentrated N-15-labe lled sample, is proposed for proteins where the expression system show s a limited efficiency.