title: 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 (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.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0307, USA.
authors.forename: Neeraj
authors.initials: N
authors.lastname: Sinha
authors.forename: Fabian V
authors.initials: FV
authors.lastname: Filipp
authors.forename: Lena
authors.initials: L
authors.lastname: Jairam
authors.forename: Sang Ho
authors.initials: SH
authors.lastname: Park
authors.forename: Joel
authors.initials: J
authors.lastname: Bradley
authors.forename: Stanley J
authors.initials: SJ
authors.lastname: Opella
mesh_terms: D001435:Bacteriophages; D002247:Carbon Isotopes; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry*; D014758:Viral Core Proteins; D015678:Viral Structural Proteins / Q000737:chemistry
chemical_list: D002247:Carbon Isotopes; D008565:Membrane Proteins; D014758:Viral Core Proteins; D015678:Viral Structural Proteins