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 labeling can be used to define inter-domain interactions in NMR structure determination. Labeling of post-translational modified proteins like glycoproteins remains difficult but some promising developments were recently achieved. Key achievements are segmental and site-specific labeling schemes that improve resonance assignment and structure determination of the glycan moiety. We adjusted the focus of this perspective article to concentrate on the NMR applications based on recent developments rather than on labeling methods themselves to illustrate the considerable potential for biomolecular NMR.
Content Type Journal Article
Pages 51-65
DOI 10.1007/s10858-009-9362-7
Authors
Lenka Skrisovska, ETH Zürich Institute for Molecular Biology and Biophysics 8093 Zurich Switzerland
Mario Schubert, ETH Zürich Institute for Molecular Biology and Biophysics 8093 Zurich Switzerland
A segmental labeling strategy for unambiguous determination of domainâ??domain interactions of large multi-domain proteins
A segmental labeling strategy for unambiguous determination of domainâ??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 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...
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[NMR paper] Segmental isotopic labeling for structural biological applications of NMR.
Segmental isotopic labeling for structural biological applications of NMR.
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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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[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.
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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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[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.
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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...
[NMR paper] Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for
Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for NMR analysis.
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J Biomol NMR. 1995 Sep;6(2):129-34
Authors: Kigawa T, Muto Y, Yokoyama S
For the application of multidimensional NMR spectroscopy to larger proteins, it would be useful to perform selective labeling of one of the 20 amino acids. For some amino acids, however, amino acid metabolism drastically reduces the efficiency and selectivity...
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Uniform 13C isotope labeling of proteins with sodium acetate for NMR studies: application to human carbonic anhydrase II.
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Biochemistry. 1991 May 7;30(18):4491-4
Authors: Venters RA, Calderone TL, Spicer LD, Fierke CA
Uniform double labeling of proteins for NMR studies can be prohibitively expensive, even with an efficient expression and purification scheme, due largely to the high cost of glucose. We...
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A simple method for amino acid selective isotope labeling of recombinant proteins in E. coli
A simple method for amino acid selective isotope labeling of recombinant proteins in E. coli
Kit I. Tong, Masayuki Yamamoto and Toshiyuki Tanaka
Journal of Biomolecular NMR; 2008; 42(1); pp 59-67
Abstract:
A simple and user-friendly method of labeling protein selectively with amino acids in vivo is introduced. This technique does not require the use of transaminase-deficient or auxotrophic strains. By manipulating the product feedback inhibitory loops of the E. coli amino acid metabolic pathways and, if necessary, by using enzyme inhibitors, proteins were labeled efficiently in vivo...
Recent advances in segmental isotope labeling of proteins: NMR applications to large proteins and glycoproteins
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