Tailoring 13 C 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 13 C labeling schemes with the purpose of overcoming the interfering effects of 13 C 13 C 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% 13 C in all sites and metabolically labeled from bacterial growth on media containing [2‐ 13 C]‐glycerol or [1,3‐ 13 C]‐glycerol enables direct 13 C detection in solid‐state NMR experiments without the need for homonuclear 13 C 13 C dipole–dipole decoupling. The 13 C‐detected NMR spectra of Pf1 coat protein show a substantial increase in sensitivity compared to the equivalent 15 N‐detected spectra. The isotopic labeling pattern was analyzed for [2‐ 13 C]‐glycerol and [1,3‐ 13 C]‐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 13 C‐labeled samples, in contrast to their failure with conventional 100% uniformly 13 C‐labeled samples. Copyright © 2007 John Wiley & Sons, Ltd.