Methyl groups as probes for proteins and complexes in in-cell NMR experiments.
Studying protein components of large intracellular complexes by in-cell NMR has so far been impossible because the backbone resonances are unobservable due to their slow tumbling rates. We describe a methodology that overcomes this difficulty through selective labeling of methyl groups, which possess more favorable relaxation behavior. Comparison of different in-cell labeling schemes with three different proteins, calmodulin, NmerA, and FKBP, shows that selective labeling with [(13)C]methyl groups on methionine and alanine provides excellent sensitivity with low background levels at very low costs.
Serber
Zach
Z
Graduate Group in Biophysics, University of California San Francisco, San Francisco, California 94143, USA.
Straub
Wesley
W
Corsini
Lorenzo
L
Nomura
Anson M
AM
Shimba
Nobuhisa
N
Craik
Charles S
CS
Ortiz de Montellano
Paul
P
Dötsch
Volker
V
D002247:Carbon Isotopes / Q000737:chemistry; D002477:Cells / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry*; D008715:Methionine / Q000737:chemistry; D008745:Methylation; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D004364:Pharmaceutical Preparations / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000737:chemistry*; D019289:Pyruvic Acid / Q000737:chemistry; D022021:Tacrolimus Binding Proteins / Q000737:chemistry
D002247:Carbon Isotopes; D004364:Pharmaceutical Preparations; D011506:Proteins; D019289:Pyruvic Acid; D008715:Methionine; D022021:Tacrolimus Binding Proteins