Unambiguous assignment of short- and long-range structural restraints by solid-state NMR spectroscopy with segmental isotope labeling

Chembiochem. 2015 Jan 2;16(1):51-4. doi: 10.1002/cbic.201402446. Epub 2014 Nov 12.

Abstract

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.

Keywords: amyloid; intein; isotopic labeling; protein structures; solid-state NMR spectroscopy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Carbon Isotopes
  • DNA Polymerase III / chemistry*
  • DNA Polymerase III / genetics
  • Gene Expression
  • Inteins / genetics*
  • Isotope Labeling
  • Models, Molecular
  • Molecular Sequence Data
  • Nitrogen Isotopes
  • Nostoc / chemistry*
  • Nostoc / genetics
  • Nuclear Magnetic Resonance, Biomolecular / methods
  • Protein Aggregates
  • Protein Conformation
  • Protein Splicing

Substances

  • Bacterial Proteins
  • Carbon Isotopes
  • Nitrogen Isotopes
  • Protein Aggregates
  • DNA Polymerase III
  • DNA polymerase III, alpha subunit