title: An extended combinatorial 15N, 13Cα, and 13C' labeling approach to protein backbone resonance assignment.
abstract: Solution NMR studies of α-helical membrane proteins are often complicated by severe spectral crowding. In addition, hydrophobic environments like detergent micelles, isotropic bicelles or nanodiscs lead to considerably reduced molecular tumbling rates which translates into line-broadening and low sensitivity. Both difficulties can be addressed by selective isotope labeling methods. In this publication, we propose a combinatorial protocol that utilizes four different classes of labeled amino acids, in which the three backbone heteronuclei (amide nitrogen, α-carbon and carbonyl carbon) are enriched in (15)N or (13)C isotopes individually as well as simultaneously. This results in eight different combinations of dipeptides giving rise to cross peaks in (1)H-(15)N correlated spectra. Their differentiation is achieved by recording a series of HN-detected 2D triple-resonance spectra. The utility of this new scheme is demonstrated with a homodimeric 142-residue membrane protein in DHPC micelles. Restricting the number of selectively labeled samples to three allowed the identification of the amino-acid type for 77 % and provided sequential information for 47 % of its residues. This enabled us to complete the backbone resonance assignment of the uniformly labeled protein merely with the help of a 3D HNCA spectrum, which can be collected with reasonable sensitivity even for relatively large, non-deuterated proteins.
authors.affiliation: Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University, Max-von-Laue-Str. 9, 60438, Frankfurt, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Löhr
authors.forename: Franz
authors.initials: F
authors.lastname: Tumulka
authors.forename: Christoph
authors.initials: C
authors.lastname: Bock
authors.forename: Rupert
authors.initials: R
authors.lastname: Abele
authors.forename: Volker
authors.initials: V
authors.lastname: Dötsch
mesh_terms: D002247:Carbon Isotopes / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D009587:Nitrogen Isotopes / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017433:Protein Structure, Secondary
chemical_list: D002247:Carbon Isotopes; D008565:Membrane Proteins; D009587:Nitrogen Isotopes