Tailoring 13C labeling for triple-resonance solid-state NMR experiments on aligned samples of proteins

In order to develop triple-resonance solid-state NMR spectroscopy of membrane proteins, we have implemented several different C-13 labeling schemes with the purpose of overcoming the interfering effects of C-13-C-13 dipole-dipole couplings in stationary samples. The membrane-bound form of the major coat protein of the filamentous bacteriophage Pf1 was used as an example of a well-characterized helical membrane protein. Aligned protein samples randomly enriched to 35% C-13 in all sites and metabolically labeled from bacterial growth on media containing [2-C-13]-glycerol or [1,3-C-13]-glycerol enables direct C-13 detection in solid-state NMR experiments without the need for homonuclear C-13-C-13 dipole-dipole decoupling. The C-13-detected NMR spectra of Pf1 coat protein show a substantial increase in sensitivity compared to the equivalent N-15-detected spectra. The isotopic labeling pattern was analyzed for [2-C-13]-glycerol and [1,3-C-13]-glycerol as metabolic precursors by solution-state NMR of micelle samples. Polarization inversion spin exchange at the magic angle (PISEMA) and other solid-state NMR experiments work well on 35% random fractionally and metabolically tailored C-13-labeled samples, in contrast to their failure with conventional 100% uniformly C-13-labeled samples. Copyright (c) 2007 John Wiley & Sons, Ltd.