title: A segmental labeling strategy for unambiguous determination of domain-domain interactions of large multi-domain proteins.
abstract: NMR structural determination of large multi-domain proteins is a challenging task due to significant spectral overlap with a particular difficulty in unambiguous identification of domain-domain interactions. Segmental labeling is a NMR strategy that allows for isotopically labeling one domain and leaves the other domain unlabeled. This significantly simplifies spectral overlaps and allows for quick identification of domain-domain interaction. Here, a novel segmental labeling strategy is presented for detection of inter-domain NOEs. To identify domain-domain interactions in human apolipoprotein E (apoE), a multi-domain, 299-residues α-helical protein, on-column expressed protein ligation was utilized to generate a segmental-labeled apoE samples in which the N-terminal (NT-) domain was (2)H(99%)/(15)N-labeled whereas the C-terminal (CT-) domain was either (15)N- or (15)N/(13)C-labeled. 3-D (15)N-edited NOESY spectra of these segmental-labeled apoE samples allow for direct observation of the inter-domain NOEs between the backbone amide protons of the NT-domain and the aliphatic protons of the CT-domain. This straightforward approach permits unambiguous identification of 78 inter-domain NOEs, enabling accurate definition of the relative positions of both the NT- and the CT-domains and determination of the NMR structure of apoE.
authors: Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201, USA.
authors: Jianglei
authors: J
authors: Chen
authors: Jianjun
authors: J
authors: Wang
mesh_terms: D001057:Apolipoproteins E / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D008958:Models, Molecular; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D054730:Protein Interaction Domains and Motifs
chemical_list: D001057:Apolipoproteins E; D009587:Nitrogen Isotopes