Extension of the HA-detection based approach: (HCA)CON(CA)H and (HCA)NCO(CA)H experiments for the main-chain assignment of intrinsically disordered proteins

摘要

Extensive resonance overlap exacerbates assignment of intrinsically disordered proteins (IDPs). This issue can be circumvented by utilizing 15N, 13C′ and 1HN spins, where the chemical shift dispersion is mainly dictated by the characteristics of consecutive amino acid residues. Especially 15N and 13C′ spins offer superior chemical shift dispersion in comparison to 13Cα and 13Cβ spins. However, HN-detected experiments suffer from exchange broadening of amide proton signals on IDPs especially under alkali conditions. To that end, we propose here two novel HA-detected experiments, (HCA)CON(CA)H and (HCA)NCO(CA)H and a new assignment protocol based on panoply of unidirectional HA-detected experiments that enable robust backbone assignment of IDPs also at high pH. The new approach was tested at pH 6.5 and pH 8.5 on cancer/testis antigen CT16, a 110-residue IDP, and virtually complete backbone assignment of CT16 was obtained by employing the novel HA-detected experiments together with the previously introduced iH(CA)NCO scheme. Remarkably, also those 10 N-terminal residues that remained unassigned in our earlier HN-detection based assignment approach even at pH 6.5 were now readily assigned. Moreover, theoretical calculations and experimental results suggest that overall sensitivity of the new experiments is also applicable to small or medium sized globular proteins that require alkaline conditions.