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

Abstract

In order to develop triple-resonance solid-state NMR spectroscopy of membrane proteins, we have implemented several different ¹³C labeling schemes with the purpose of overcoming the interfering effects of ¹³C¹³C 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% ¹³C in all sites and metabolically labeled from bacterial growth on media containing [2-¹³C]-glycerol or [1,3-¹³C]-glycerol enables direct ¹³C detection in solid-state NMR experiments without the need for homonuclear ¹³C¹³C dipole–dipole decoupling. The ¹³C-detected NMR spectra of Pf1 coat protein show a substantial increase in sensitivity compared to the equivalent ¹⁵N-detected spectra. The isotopic labeling pattern was analyzed for [2-¹³C]-glycerol and [1,3-¹³C]-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 ¹³C-labeled samples, in contrast to their failure with conventional 100% uniformly ¹³C-labeled samples. Copyright © 2007 John Wiley & Sons, Ltd.