[NMR paper] Towards complete polypeptide backbone NH assignment via combinatorial labeling

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Towards complete polypeptide backbone NH assignment via combinatorial labeling

Publication date: Available online 29 March 2019

Source: Journal of Magnetic Resonance

Author(s): Frank Löhr, Jakob Gebel, Erik Henrich, Christopher Hein, Volker Dötsch

Abstract

Combinatorial selective isotope labeling is a valuable tool to facilitate polypeptide backbone resonance assignment in cases of low sensitivity or extensive chemical shift degeneracy. It involves recording of 15N-HSQC and 2D HN-projections of triple-resonance spectra on a limited set of samples containing different combinations of labeled and unlabeled amino acid types. Using labeling schemes in which the three backbone heteronuclei (amide nitrogen, ?-carbon and carbonyl carbon) are enriched in 15N or 13C isotopes – individually as well as simultaneously – usually yields abundant amino-acid type information of consecutive residues i and i-1. Although this results in a large number of anchor points that can be used in the sequential assignment process, for most amide signals the exact positioning of the corresponding residue the polypeptide sequence still relies on matching intra- and interresidual 13C chemical shifts obtained from 3D spectra. An obvious way to obtain more sequence-specific assignments directly with combinatorial labeling would be to increase the number of samples. This is, however, undesirable because of increased sample preparation efforts and costs. Irrespective of the number of samples, unambiguous assignments cannot be accomplished for i-1/i pairs that are not unique in the sequence. Here we show that the ambiguity for non-unique pairs can be resolved by including information about the labeling state of residues i+1 and i-2. Application to a 35-residue peptide resulted in complete assignments of all detectable signals in the 15N HSQC which, due to its repetitive sequence and 13C chemical shift degeneracies, was difficult to achieve by other means. For a medium-sized protein (165 residues, rotational correlation time 8.2 ns) the improved protocol allowed the extent of backbone amide assignment to be expanded to 88 % solely using a suite of 2D 1H-15N correlated spectra.



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