Tailoring 13C labeling for triple-resonance solid-state \NMR\ experiments on aligned samples of proteins
Magn Reson Chem, (December 2007)DOI: 10.1002/mrc.2121
Abstract
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.