title: Application of amino acid type-specific 1H- and 14N-labeling in a 2H-, 15N-labeled background to a 47 kDa homodimer: potential for NMR structure determination of large proteins.
abstract: NMR investigations of larger macromolecules (> 20 kDa) are severely hindered by rapid 1H and 13C transverse relaxation. Replacement of non-exchangeable protons with deuterium removes many efficient 1H-1H and 1H-13C relaxation pathways. The main disadvantage of deuteration is that many of the protons which would normally be the source of NOE-based distance restraints are removed. We report the development of a novel labeling strategy which is based on specific protonation and 14N-labeling of the residues phenylalanine, tyrosine, threonine, isoleucine and valine in a fully deuterated, 15N-labeled background. This allows the application of heteronuclear half-filters, 15N-editing and 1H-TOCSY experiments to select for particular magnetization transfer pathways. Results from investigations of a 47 kDa dimeric protein labeled in this way demonstrated that the method provides useful information for the structure determination of large proteins.
authors.affiliation: Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany. kelly@fmp-berlin.de
authors.forename: M J
authors.initials: MJ
authors.lastname: Kelly
authors.forename: C
authors.initials: C
authors.lastname: Krieger
authors.forename: L J
authors.initials: LJ
authors.lastname: Ball
authors.forename: Y
authors.initials: Y
authors.lastname: Yu
authors.forename: G
authors.initials: G
authors.lastname: Richter
authors.forename: P
authors.initials: P
authors.lastname: Schmieder
authors.forename: A
authors.initials: A
authors.lastname: Bacher
authors.forename: H
authors.initials: H
authors.lastname: Oschkinat
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids; D003903:Deuterium; D019281:Dimerization; D006859:Hydrogen; D008970:Molecular Weight; D009584:Nitrogen; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D009842:Oligopeptides / Q000737:chemistry*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*
chemical_list: D000596:Amino Acids; D009587:Nitrogen Isotopes; D009842:Oligopeptides; D011506:Proteins; D006859:Hydrogen; D003903:Deuterium; D009584:Nitrogen