Assigning large proteins in the solid state: a MAS NMR resonance assignment strategy using selectively and extensively C-13-labelled proteins

Victoria A. Higman, Jeremy Flinders, Matthias Hiller, Stefan Jehle, Stefan Markovic, Sebastian Fiedler, Barth-Jan van Rossum, Hartmut Oschkinat*

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)

98 Citations (Scopus)

Abstract

In recent years, solid-state magic-angle spinning nuclear magnetic resonance spectroscopy (MAS NMR) has been growing into an important technique to study the structure of membrane proteins, amyloid fibrils and other protein preparations which do not form crystals or are insoluble. Currently, a key bottleneck is the assignment process due to the absence of the resolving power of proton chemical shifts. Particularly for large proteins (approximately > 150 residues) it is difficult to obtain a full set of resonance assignments. In order to address this problem, we present an assignment method based upon samples prepared using [1,3-C-13]- and [2-C-13]-glycerol as the sole carbon source in the bacterial growth medium (so-called selectively and extensively labelled protein). Such samples give rise to higher quality spectra than uniformly [C-13]-labelled protein samples, and have previously been used to obtain long-range restraints for use in structure calculations. Our method exploits the characteristic cross-peak patterns observed for the different amino acid types in C-13-C-13 correlation and 3D NCACX and NCOCX spectra. An in-depth analysis of the patterns and how they can be used to aid assignment is presented, using spectra of the chicken alpha-spectrin SH3 domain (62 residues), alpha B-crystallin (175 residues) and outer membrane protein G (OmpG, 281 residues) as examples. Using this procedure, over 90% of the C alpha, C beta, C' and N resonances in the core domain of alpha B-crystallin and around 73% in the flanking domains could be assigned (excluding 24 residues at the extreme termini of the protein).

Original languageEnglish
Pages (from-to)245-260
Number of pages16
JournalJournal of Biomolecular NMR
Volume44
Issue number4
DOIs
Publication statusPublished - Aug 2009

Keywords

  • Isotopic labelling
  • CONFORMATIONAL-ANALYSIS
  • ROTATING SOLIDS
  • CORRELATION SPECTROSCOPY
  • ESCHERICHIA-COLI THIOREDOXIN
  • NUCLEAR-MAGNETIC-RESONANCE
  • Membrane proteins
  • PERDEUTERATED PROTEINS
  • CHEMICAL-SHIFT ASSIGNMENTS
  • MICROCRYSTALLINE UBIQUITIN
  • Resonance assignment
  • ANGLE-SPINNING NMR
  • SIDE-CHAIN C-13
  • Solid-state NMR
  • Protein structure determination

Cite this

Higman, V. A., Flinders, J., Hiller, M., Jehle, S., Markovic, S., Fiedler, S., van Rossum, B-J., & Oschkinat, H. (2009). Assigning large proteins in the solid state: a MAS NMR resonance assignment strategy using selectively and extensively C-13-labelled proteins. Journal of Biomolecular NMR, 44(4), 245-260. https://doi.org/10.1007/s10858-009-9338-7