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.
Abstract13 C-detected solid-state NMR experiments have substantially higher sensitivity than the corresponding 15 N-detected experiments on stationary, aligned samples of isotopically labeled proteins. Several methods for tailoring the isotopic labeling are described that result in spatially isolated 13 C sites so that dipole-dipole couplings among the 13 C are minimized, thus eliminating the need for homonuclear 13 C-13 C decoupling in either indirect or direct dimensions of one-or multidimensional NMR experiments that employ 13 C detection. The optimal percentage for random fractional 13 C labeling is between 25% and 35%. Specifically labeled glycerol and glucose can be used at the carbon sources to tailor the isotopic labeling, and the choice depends on the resonances of interest for a particular study. For investigations of the protein backbone, growth of the bacteria on 2-13 C-glucose containing media was found to be most effective.
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