Abstract
Spectral projection experiments by NMR in conjunction with decomposition analysis have been previously introduced for the backbone assignment of proteins; various pulse sequences as well as the behaviour with low signal-to-noise or chemical shift degeneracy have been illustrated. As a guide for routine applications of this combined tool, we provide here a systematic analysis on different types of proteins using welldefined run-time parameters. As a second result of this study, the backbone assignment module SHABBA was extensively rewritten and improved. Calculations on ubiquitin yielded again fully correct and nearly complete backbone and CHβ assignments. For the 128 residue long azurin, missing assignments mostly affect Hα and Hβ. Among the remaining backbone (plus Cβ) nuclei 97.5 % could be assigned with 1.0 % differences to a reference. Finally, the new SHABBA algorithm was applied to projections recorded for a yeast histone protein domain at room temperature, where the protein is subject to partial unfolding: this leads to unobservable resonances (about a dozen missing signals in a normal 15N-HSQC) and extensive degeneracy among the resonances. From the clearly observable residues, 97.5 % of the backbone and CHβresonances could be assigned, of which only 0.8 % showed differences to published shifts. An additional study on the protein MMP20, which exhibits spectral difficulties to an even larger extent, explores the limitations of the approach.
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Acknowledgments
This work was supported by the Swedish NMR Centre and the EU ExtendNMR project. JF thanks the Lawski Foundation for financial support. Data for MMP20 were recorded at CERM (support by the EU-NMR European Network of Research Infrastructures). We thank Tim Stevens and CERM for loans of NMR samples with the histone domain and MMP20, respectively.
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Fredriksson, J., Bermel, W., Staykova, D.K. et al. Automated protein backbone assignment using the projection-decomposition approach. J Biomol NMR 54, 43–51 (2012). https://doi.org/10.1007/s10858-012-9649-y
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DOI: https://doi.org/10.1007/s10858-012-9649-y