Selective and extensive 13C labeling of a membrane protein for solid-state NMR investigations.
The selective and extensive 13C labeling of mostly hydrophobic amino acid residues in a 25 kDa membrane protein, the colicin Ia channel domain, is reported. The novel 13C labeling approach takes advantage of the amino acid biosynthetic pathways in bacteria and suppresses the synthesis of the amino acid products of the citric acid cycle. The selectivity and extensiveness of labeling significantly simplify the solid-state NMR spectra, reduce line broadening, and should permit the simultaneous measurement of multiple structural constraints. We show the assignment of most 13C resonances to specific amino acid types based on the characteristic chemical shifts, the 13C labeling pattern, and the amino acid composition of the protein. The assignment is partly confirmed by a 2D homonuclear double-quantum-filter experiment under magic-angle spinning. The high sensitivity and spectral resolution attained with this 13C-labeling protocol, which is termed TEASE for ten-amino acid selective and extensive labeling, are demonstrated.
Hong
M
M
Department of Polymer Science & Engineering, University of Massachusetts, Amherst 01003, USA. mhong@chem.umass.edu
Jakes
K
K
D000595:Amino Acid Sequence; D002247:Carbon Isotopes; D003087:Colicins / Q000737:chemistry*; D007473:Ion Channels; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D017433:Protein Structure, Secondary; D011789:Quantum Theory
D002247:Carbon Isotopes; D003087:Colicins; D007473:Ion Channels; D008565:Membrane Proteins