title: Automated assignment in selectively methyl-labeled proteins.
abstract: Specific methyl labeling schemes and transverse relaxation optimized spectroscopy (TROSY) has extended the molecular size range for the application of NMR spectroscopy to very large proteins (up to approximately 1 MDa). Existing strategies for resonance assignment of methyl groups in large systems are based on NMR spectra recorded on smaller fragments and mutants. This is very time-consuming, and chemical shift changes due to mutation or truncation can often complicate interpretation. We have developed a new automated procedure able to rapidly assign the majority of methyl groups in very large proteins, without recourse to mutagenesis or truncated fragments (http://nmr.bc.ic.ac.uk/map-xs/). We demonstrate the effectiveness of this approach on the 300 kDa, ILV-labeled proteasome (alpha(7)alpha(7)) for which excellent spectra have been previously recorded. Of the observed methyl groups, 99% can be correctly assigned in a matter of minutes without manual intervention.
authors.affiliation: Division of Molecular Biosciences, Cross Faculty NMR Centre, Imperial College London, South Kensington, London, SW7 2AZ, UK.
authors.forename: Yingqi
authors.initials: Y
authors.lastname: Xu
authors.forename: Minhao
authors.initials: M
authors.lastname: Liu
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Simpson
authors.forename: Rivka
authors.initials: R
authors.lastname: Isaacson
authors.forename: Ernesto
authors.initials: E
authors.lastname: Cota
authors.forename: Jan
authors.initials: J
authors.lastname: Marchant
authors.forename: Daiwen
authors.initials: D
authors.lastname: Yang
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Zhang
authors.forename: Paul
authors.initials: P
authors.lastname: Freemont
authors.forename: Stephen
authors.initials: S
authors.lastname: Matthews
mesh_terms: D001331:Automation; D018360:Crystallography, X-Ray; D003903:Deuterium / Q000737:chemistry*; D008745:Methylation; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011506:Proteins / Q000737:chemistry*
chemical_list: D011506:Proteins; D003903:Deuterium