Unambiguous assignment of short- and long-range structural restraints by solid-state NMR spectroscopy with segmental isotope labeling.
We present an efficient method for the reduction of spectral complexity in the solid-state NMR spectra of insoluble protein assemblies, without loss of signal intensity. The approach is based on segmental isotope labeling by using the split intein DnaE from Nostoc punctiforme. We show that the segmentally (13)C, (15)N-labeled prion domain of HET-s exhibits significantly reduced spectral overlap while retaining the wild-type structure and spectral quality. A large number of unambiguous distance restraints were thus collected from a single two-dimensional (13)C, (13)C cross-correlation spectrum. The observed resonances could be unambiguously identified as intramolecular without the need for preparing a dilute, less sensitive sample.
Laboratory of Macromolecular Interactions, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig (Germany).
Tobias
T
Schubeis
Thorsten
T
Lührs
Christiane
C
Ritter
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002247:Carbon Isotopes; D004258:DNA Polymerase III / Q000737:chemistry* / Q000235:genetics; D015870:Gene Expression; D047668:Inteins / Q000235:genetics*; D007553:Isotope Labeling; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D046937:Nostoc / Q000737:chemistry* / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D066329:Protein Aggregates; D011487:Protein Conformation; D019154:Protein Splicing
D001426:Bacterial Proteins; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D066329:Protein Aggregates; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III
amyloid; intein; isotopic labeling; protein structures; solid-state NMR spectroscopy