Selective and extensive 13C labeling of a membrane protein for solid-state NMR investigations

The selective and extensive C-13 labeling of mostly hydrophobic amino acid residues in a 25 kDa membrane protein, the colicin Ia channel domain, is reported. The novel C-13 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 C-13 resonances to specific amino acid types based on the characteristic chemical shifts, the C-13 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 C-13-labeling protocol, which is termed TEASE for ten-amino acid selective and extensive labeling, are demonstrated.