A practical implementation of cross-spectrum in protein backbone resonance assignment

The concept of cross-spectrum is applied in protein NMR spectroscopy to assist in the backbone sequential resonance assignment. Cross-spectrum analysis is used routinely to reveal correlations in frequency domains as a means to reveal common features contained in multiple time series. Here the cross-spectrum between related NMR spectra, for example HNCO and HN(CA)CO, can be calculated with point-by-point multiplications along their common C′ carbon axis. In the resulting higher order cross-spectrum, an enhanced correlation signal occurs at every common i-1 carbon frequency allowing the amide proton HN (and nitrogen N) resonances from residues i and i-1 to be identified. The cross-spectrum approach is demonstrated using 2D spectra H(N)CO, H(NCA)CO, H(NCO)CACB, and H(N)CACB measured on a 15N/13C double-labeled Ubiquitin sample. These 2D spectra are used to calculate two pseudo-3D cross-spectra, Hi-Hi-1-C′i-1 and Hi-Hi-1-CAi-1CBi-1. We show using this approach, backbone resonances of H, C′, CA, and CB can be fully assigned without ambiguity. The cross-spectrum principle is expected to offer an easy, practical, and more quantitative approach for heteronuclear backbone resonance assignment.