Automated assignment in selectively methyl-labeled proteins.
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.
Xu
Yingqi
Y
Division of Molecular Biosciences, Cross Faculty NMR Centre, Imperial College London, South Kensington, London, SW7 2AZ, UK.
Liu
Minhao
M
Simpson
Peter J
PJ
Isaacson
Rivka
R
Cota
Ernesto
E
Marchant
Jan
J
Yang
Daiwen
D
Zhang
Xiaodong
X
Freemont
Paul
P
Matthews
Stephen
S
D001331:Automation; D018360:Crystallography, X-Ray; D003903:Deuterium / Q000737:chemistry*; D008745:Methylation; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011506:Proteins / Q000737:chemistry*
D011506:Proteins; D003903:Deuterium