title: Protocols for the sequential solid-state NMR spectroscopic assignment of a uniformly labeled 25 kDa protein: HET-s(1-227).
abstract: The sequence-specific resonance assignment of a protein forms the basis for studies of molecular structure and dynamics, as well as to functional assay studies by NMR spectroscopy. Here we present a protocol for the sequential 13C and 15N resonance assignment of uniformly [15N,13C]-labeled proteins, based on a suite of complementary three-dimensional solid-state NMR spectroscopy experiments. It is directed towards the application to proteins with more than about 100 amino acid residues. The assignments rely on a walk along the backbone by using a combination of three experiments that correlate nitrogen and carbon spins, including the well-dispersed Cbeta resonances. Supplementary spectra that correlate further side-chain resonances can be important for identifying the amino acid type, and greatly assist the assignment process. We demonstrate the application of this assignment protocol for a crystalline preparation of the N-terminal globular domain of the HET-s prion, a 227-residue protein.
authors.affiliation: Physical Chemistry, ETH Zürich, 8093 Zürich, Switzerland.
authors.forename: Anne
authors.initials: A
authors.lastname: Schuetz
authors.forename: Christian
authors.initials: C
authors.lastname: Wasmer
authors.forename: Birgit
authors.initials: B
authors.lastname: Habenstein
authors.forename: René
authors.initials: R
authors.lastname: Verel
authors.forename: Jason
authors.initials: J
authors.lastname: Greenwald
authors.forename: Roland
authors.initials: R
authors.lastname: Riek
authors.forename: Anja
authors.initials: A
authors.lastname: Böckmann
authors.forename: Beat H
authors.initials: BH
authors.lastname: Meier
mesh_terms: D000595:Amino Acid Sequence; D002247:Carbon Isotopes; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009587:Nitrogen Isotopes; D011328:Prions / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D012107:Research Design
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011328:Prions; D011506:Proteins