Proton-detected MAS NMR experiments based on dipolar transfers for backbone assignment of highly deuterated proteins

J Magn Reson. 2014 May:242:180-8. doi: 10.1016/j.jmr.2014.02.020. Epub 2014 Mar 4.

Abstract

Proton-detected solid-state NMR was applied to a highly deuterated insoluble, non-crystalline biological assembly, the Salmonella typhimurium type iii secretion system (T3SS) needle. Spectra of very high resolution and sensitivity were obtained at a low protonation level of 10-20% at exchangeable amide positions. We developed efficient experimental protocols for resonance assignment tailored for this system and the employed experimental conditions. Using exclusively dipolar-based interspin magnetization transfers, we recorded two sets of 3D spectra allowing for an almost complete backbone resonance assignment of the needle subunit PrgI. The additional information provided by the well-resolved proton dimension revealed the presence of two sets of resonances in the N-terminal helix of PrgI, while in previous studies employing (13)C detection only a single set of resonances was observed.

Keywords: Deuteration; Magic-angle spinning solid-state NMR; PrgI; Protein resonance assignment; Proton detection; Type 3 secretion system.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms*
  • Amino Acid Sequence
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / ultrastructure*
  • Computer Simulation
  • Deuterium / chemistry*
  • Deuterium Exchange Measurement / methods
  • Models, Chemical
  • Molecular Sequence Data
  • Proton Magnetic Resonance Spectroscopy / methods*
  • Salmonella typhimurium / metabolism*

Substances

  • Bacterial Proteins
  • Deuterium