High-resolution 3D HNCOCA experiment applied to a 28 kDa paramagnetic protein

A new triple-resonance 3D HNCOCA pulse scheme is presented, designed to identify the backbone nuclei (H(N), N, CO, C(α)) of doubly labelled proteins. The two carbon frequencies are labelled along the same indirect 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(α). If one takes advantage of the symmetry properties of the spectra in the course of the peak picking, this 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 protein of 2×14 kDa. A resonance assignment strategy, based on a low-concentration (13)C/(15)N-labelled sample and a more concentrated (15)N-labelled sample, is proposed for proteins where the expression system shows a limited efficiency.