A segmental labeling strategy for unambiguous determination of domain–domain interactions of large multi-domain proteins

• Published in: Journal of biomolecular NMR Vol. 50; no. 4; pp. 403 - 410
• Main Authors: Chen, Jianglei, Wang, Jianjun
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Netherlands 01.08.2011; Springer Nature B.V
• Subjects: Apolipoproteins E - chemistry; Apolipoproteins E - metabolism; Binding Sites; Biochemistry; Biological and Medical Physics; Biophysics; Humans; Isotope Labeling - methods; Models, Molecular; Nitrogen Isotopes; Nuclear Magnetic Resonance, Biomolecular - methods; Physics; Physics and Astronomy; Protein Interaction Domains and Motifs; Spectroscopy/Spectrometry; NMR; NOESY; Inter-domain interaction; Segmental labeling; NOE; apoE3
• ISSN: 0925-2738; 1573-5001
• DOI: 10.1007/s10858-011-9526-0

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.