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 2H(99%)/15N-labeled whereas the C-terminal (CT-) domain was either 15N- or 15N/13C-labeled. 3-D 15N-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.
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Acknowledgments
This work is supported by NIH RO1 grant from the NIH (HL074365 to Jianjun Wang), a grant from the American Health Assistant Foundation (Jianjun Wang). The authors also thank Rebecca Wang for critical reading the manuscript.
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Chen, J., Wang, J. A segmental labeling strategy for unambiguous determination of domain–domain interactions of large multi-domain proteins. J Biomol NMR 50, 403–410 (2011). https://doi.org/10.1007/s10858-011-9526-0
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DOI: https://doi.org/10.1007/s10858-011-9526-0