Here we introduce a new pulse sequence for resonance assignment that halves the number of data sets required for sequential linking by directly correlating sequential amide resonances in a single diagonal-free spectrum. The method is demonstrated with both microcrystalline and sedimented deuterated proteins spinning at 60 and 111 kHz, and a fully protonated microcrystalline protein spinning at 111 kHz, with as little as 0.5 mg protein sample. We find that amide signals have a low chance of ambiguous linkage, which is further improved by linking in both forward and backward directions. The spectra obtained are amenable to automated resonance assignment using general-purpose software such as UNIO-MATCH.
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We thank the members of the technical staff of the ISA for assistance with the NMR spectrometers. We acknowledge support from CNRS (Fondation pour la Chimie des Substances Naturelles) and from the People Programme of the European Union’s FP7 (FP7-PEOPLE-2012-ITN REA Grant agreement No 317127 “pNMR” and 316630 “CAS-IDP”). LBA is supported by a MC incoming fellowship (REA Grant agreement No 624918 “MEM-MAS”), and JS by an EMBO fellowship (ALTF 1506-2014) and by the Marie Curie Actions of the European Commission (LTFCOFUND2013, GA-2013-609409).
Electronic supplementary material
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Sample preparation, comparison of 1H, 13C and 15N resolution and degree of overlap for inter-residue correlation, simulated 13C-13C transfer efficiency curves. Supplementary material 1 (PDF 985 kb)
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Andreas, L.B., Stanek, J., Le Marchand, T. et al. Protein residue linking in a single spectrum for magic-angle spinning NMR assignment. J Biomol NMR 62, 253–261 (2015). https://doi.org/10.1007/s10858-015-9956-1
- Magic-angle spinning
- Protein resonance assignment
- Proton detection