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 domainâ??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 domainâ??domain interaction. Here, a novel segmental labeling strategy is presented for detection of inter-domain NOEs. To identify domainâ??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.
Content Type Journal Article
Pages 1-8
DOI 10.1007/s10858-011-9526-0
Authors
Jianglei Chen, Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201, USA
Jianjun Wang, Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201, USA
Insight into interactions of the von-Willebrand-factor-A-like domain 2 with the FNIII-like domain 9 of collagen VII by NMR and SPR.
Insight into interactions of the von-Willebrand-factor-A-like domain 2 with the FNIII-like domain 9 of collagen VII by NMR and SPR.
Insight into interactions of the von-Willebrand-factor-A-like domain 2 with the FNIII-like domain 9 of collagen VII by NMR and SPR.
FEBS Lett. 2011 May 9;
Authors: Leineweber S, Schönig S, Seeger K
Type VII collagen as component of anchoring fibrils plays an important role in skin architecture, however, no detailed structural information is available. Here, we describe the recombinant expression, isotope labeling, and...
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05-17-2011 06:21 PM
Recent advances in segmental isotope labeling of proteins: NMR applications to large proteins and glycoproteins
Recent advances in segmental isotope labeling of proteins: NMR applications to large proteins and glycoproteins
Abstract In the last 15 years substantial advances have been made to place isotope labels in native and glycosylated proteins for NMR studies and structure determination. Key developments include segmental isotope labeling using Native Chemical Ligation, Expressed Protein Ligation and Protein Trans-Splicing. These advances are pushing the size limit of NMR spectroscopy further making larger proteins accessible for this technique. It is just emerging that segmental isotope...
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01-09-2011 12:46 PM
Observing selected domains in multi-domain proteins via sortase-mediated ligation and NMR spectroscopy
Observing selected domains in multi-domain proteins via sortase-mediated ligation and NMR spectroscopy
Abstract NMR spectroscopy has distinct advantages for providing insight into protein structures, but faces significant resolution challenges as protein size increases. To alleviate such resonance overlap issues, the ability to produce segmentally labeled proteins is beneficial. Here we show that the S. aureus transpeptidase sortase A can be used to catalyze the ligation of two separately expressed domains of the same protein, MecA (B. subtilis). The yield of purified, segmentally...
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12-31-2010 08:38 PM
Observing selected domains in multi-domain proteins via sortase-mediated ligation and NMR spectroscopy.
Observing selected domains in multi-domain proteins via sortase-mediated ligation and NMR spectroscopy.
Observing selected domains in multi-domain proteins via sortase-mediated ligation and NMR spectroscopy.
J Biomol NMR. 2010 Dec 29;
Authors: Refaei MA, Combs A, Kojetin DJ, Cavanagh J, Caperelli C, Rance M, Sapitro J, Tsang P
NMR spectroscopy has distinct advantages for providing insight into protein structures, but faces significant resolution challenges as protein size increases. To alleviate such resonance overlap issues, the ability to...
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12-29-2010 04:04 PM
[NMR paper] Folding and domain-domain interactions of the chaperone PapD measured by 19F NMR.
Folding and domain-domain interactions of the chaperone PapD measured by 19F NMR.
Related Articles Folding and domain-domain interactions of the chaperone PapD measured by 19F NMR.
Biochemistry. 2004 Nov 2;43(43):13775-86
Authors: Bann JG, Frieden C
The folding of the two-domain bacterial chaperone PapD has been studied to develop an understanding of the relationship between individual domain folding and the formation of domain-domain interactions. PapD contains six phenylalanine residues, four in the N-terminal domain and two in the...
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11-24-2010 10:03 PM
[NMR paper] Segmental isotopic labeling for structural biological applications of NMR.
Segmental isotopic labeling for structural biological applications of NMR.
Related Articles Segmental isotopic labeling for structural biological applications of NMR.
Methods Mol Biol. 2004;278:47-56
Authors: Cowburn D, Shekhtman A, Xu R, Ottesen JJ, Muir TW
This chapter describes the preparation of precursor domains for the formation of multidomain segmentally labeled proteins by protein ligation.
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11-24-2010 09:25 PM
[NMR paper] Chain-selective isotopic labeling for NMR studies of large multimeric proteins: appli
Chain-selective isotopic labeling for NMR studies of large multimeric proteins: application to hemoglobin.
Related Articles Chain-selective isotopic labeling for NMR studies of large multimeric proteins: application to hemoglobin.
Biophys J. 2000 Aug;79(2):1146-54
Authors: Simplaceanu V, Lukin JA, Fang TY, Zou M, Ho NT, Ho C
Multidimensional, multinuclear NMR has the potential to elucidate the mechanisms of allostery and cooperativity in multimeric proteins under near-physiological conditions. However, NMR studies of proteins made up of...
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11-19-2010 08:29 PM
[NMR paper] Chemical ligation of folded recombinant proteins: segmental isotopic labeling of doma
Chemical ligation of folded recombinant proteins: segmental isotopic labeling of domains for NMR studies.
Related Articles Chemical ligation of folded recombinant proteins: segmental isotopic labeling of domains for NMR studies.
Proc Natl Acad Sci U S A. 1999 Jan 19;96(2):388-93
Authors: Xu R, Ayers B, Cowburn D, Muir TW
A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology...
A segmental labeling strategy for unambiguous determination of domainâ??domain interactions of large multi-domain proteins
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