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
- 245 Downloads
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.
KeywordsAssignment CT16 HA-detection (HCA)CON(CA)H (HCA)NCO(CA)H Intrinsically disordered proteins IDP
This work was financially supported by the grants 122170 and 131144 (to P. P.) from the Academy of Finland. Sampo Mäntylahti acknowledges The National Doctoral programme in Informational and Structural Biology (ISB).
- Goddard T, Kneller D (2004) Sparky 3. University of California, San FranciscoGoogle Scholar
- Kay LE, Ikura M, Tschudin R, Bax A (1990) Three-dimensional triple-resonance NMR spectroscopy of isotopically enriched proteins. J Magn Reson 89:496–514Google Scholar
- Kupće E, Wagner G (1995) Wideband homonuclear decoupling in protein spectra. J Magn Reson 109A:329–333Google Scholar
- Marion D, Ikura M, Tschudin R, Bax A (1989) Rapid recording of 2D NMR-spectra without phase cycling—application to the study of hydrogen-exchange in proteins. J Magn Reson 85:393–399Google Scholar
- Matsuo H, Kupce E, Li H, Wagner G (1996) Use of selective Cα pulses for improvement of HN(CA)CO-D and HN(COCA)NH-D experiments. J Magn Reson 111B:194–198Google Scholar
- Pervushin K, Riek R, Wider G, Wüthrich K (1997) Attenuated T2 relaxation by mutual cancellation of dipole–dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci USA 94:12366–12731CrossRefADSGoogle Scholar
- Rappu P, Nylund C, Ristiniemi N, Kulpakko J, Vihinen P, Hernberg M, Mirtti T, Alanen K, Kallajoki M, Vuoristo M-S, Pyrhönen S, Heino J (2010) Detection of melanoma-derived cancer–testis antigen CT16 in patient sera by a novel immunoassay. Int J Cancer. doi: 10.1002/ijc.25571
- Shaka AJ, Keeler J, Frenkiel T, Freeman R (1983) An improved sequence for broad-band decoupling—Waltz-16. J Magn Reson 52:335–338Google Scholar