ausblenden:
Schlagwörter:
Solid-state NMR; Isotopic labeling; Resonance assignment; Structure determination; Secretion systems; Supramolecular assemblies
Zusammenfassung:
13C spin diluted protein samples can be produced using [1-13C] and [2-13C]-glucose (Glc) carbon sources in the bacterial growth medium. The 13C spin dilution results in favorable 13C spectral resolution and polarization transfer behavior. We recently reported the combined use of [1-13C]- and [2-13C]-Glc labeling to facilitate the structural analysis of insoluble and non-crystalline biological systems by solid-state NMR (ssNMR), including sequential assignment, detection of long-range contacts and structure determination of macromolecular assemblies. In solution NMR the beneficial properties of sparsely labeled samples using [2-13C]-glycerol (13C labeled Cα sites on a 12C diluted background) have recently been exploited to provide a bi-directional assignment method (Takeuchi et al. in J Biomol NMR 49(1):17–26, 2011 ). Inspired by this approach and our own recent results using [2-13C]-Glc as carbon sources for the simplification of ssNMR spectra, we present a strategy for a bi-directional sequential assignment of solid-state NMR resonances and additionally the detection of long-range contacts using the combination of 13C spin dilution and 3D NMR spectroscopy. We illustrate our results with the sequential assignment and the collection of distance restraints on an insoluble and non-crystalline supramolecular assembly, the Salmonella typhimurium type III secretion system needle.
