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Default Segmental isotope labelling and solid-state NMR of a 12â??Ã?â??59Â*kDa motor protein: identification of structural variability

Segmental isotope labelling and solid-state NMR of a 12â??Ã?â??59Â*kDa motor protein: identification of structural variability

Abstract

Segmental isotope labelling enables the NMR study of an individual domain within a multidomain protein, but still in the context of the entire full-length protein. Compared to the fully labelled protein, spectral overlap can be greatly reduced. We here describe segmental labelling of the (double-) hexameric DnaB helicase from Helicobacter pylori using a ligation approach. Solid-state spectra demonstrate that the ligated protein has the same structure and structural order as the directly expressed full-length protein. We uniformly 13C/15N labeled the N-terminal domain (147 residues) of the protein, while the C-terminal domain (311 residues) remained in natural abundance. The reduced signal overlap in solid-state NMR spectra allowed to identify structural â??hotspotsâ?? for which the structure of the N-terminal domain in the context of the oligomeric full-length protein differs from the one in the isolated form. They are located near the linker between the two domains, in an α-helical hairpin.



Source: Journal of Biomolecular NMR
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