title: Increased resolution of aromatic cross peaks using alternate 13C labeling and TROSY.
abstract: For typical globular proteins, contacts involving aromatic side chains would constitute the largest number of distance constraints that could be used to define the structure of proteins and protein complexes based on NOE contacts. However, the (1)H NMR signals of aromatic side chains are often heavily overlapped, which hampers extensive use of aromatic NOE cross peaks. Some of this overlap can be overcome by recording (13)C-dispersed NOESY spectra. However, the resolution in the carbon dimension is rather low due to the narrow dispersion of the carbon signals, large one-bond carbon-carbon (C-C) couplings, and line broadening due to chemical shift anisotropy (CSA). Although it has been noted that the CSA of aromatic carbons could be used in TROSY experiments for enhancing resolution, this has not been used much in practice because of complications arising from large aromatic one-bond C-C couplings, and 3D or 4D carbon dispersed NOESY are typically recorded at low resolution hampering straightforward peak assignments. Here we show that the aromatic TROSY effect can optimally be used when employing alternate (13)C labeling using 2-(13)C glycerol, 2-(13)C pyruvate, or 3-(13)C pyruvate as the carbon source. With the elimination of the strong one-bond C-C coupling, the TROSY effect can easily be exploited. We show that (1)H-(13)C TROSY spectra of alternately (13)C labeled samples can be recorded at high resolution, and we employ 3D NOESY aromatic-TROSY spectra to obtain valuable intramolecular and intermolecular cross peaks on a protein complex.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA, 02115, USA.
authors.forename: Alexander G
authors.initials: AG
authors.lastname: Milbradt
authors.forename: Haribabu
authors.initials: H
authors.lastname: Arthanari
authors.forename: Koh
authors.initials: K
authors.lastname: Takeuchi
authors.forename: Andras
authors.initials: A
authors.lastname: Boeszoermenyi
authors.forename: Franz
authors.initials: F
authors.lastname: Hagn
authors.forename: Gerhard
authors.initials: G
authors.lastname: Wagner
mesh_terms: D024322:Amino Acids, Aromatic / Q000737:chemistry*; D002247:Carbon Isotopes / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000737:chemistry*
chemical_list: D024322:Amino Acids, Aromatic; D002247:Carbon Isotopes; D011506:Proteins