Segmental isotopic labeling can facilitate NMR studies of large proteins, multi-domain proteins, and proteins with repetitive sequences by alleviating NMR signal overlaps. Segmental isotopic labeling also allows us to investigate an individual domain in the context of a full-length protein by NMR. Several established methods are available for segmental isotopic labeling such as intein-mediated ligation, but each has specific requirements and limitations. Here, we report an enzymatic approach using bacterially produced asparagine endopeptidase from Oldenlandia affinis for segmental isotopic labeling of a protein with repetitive sequences, a designed armadillo repeat protein, by overcoming some of the shortcomings of enzymatic ligation for segmental isotopic labeling.
[NMR paper] Segmental isotopic labeling of HIV-1 capsid protein assemblies for solid state NMR.
Segmental isotopic labeling of HIV-1 capsid protein assemblies for solid state NMR.
Related Articles Segmental isotopic labeling of HIV-1 capsid protein assemblies for solid state NMR.
J Biomol NMR. 2018 Jan 18;:
Authors: Gupta S, Tycko R
Abstract
Recent studies of noncrystalline HIV-1 capsid protein (CA) assemblies by our laboratory and by Polenova and coworkers (Protein Sci 19:716-730, 2010; J Mol Biol 426:1109-1127, 2014; J Biol Chem 291:13098-13112, 2016; J Am Chem Soc 138:8538-8546, 2016; J Am Chem Soc 138:12029-12032, 2016;...
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02-23-2018 03:44 AM
Segmental isotopic labeling of HIV-1 capsid protein assemblies for solid state NMR
Segmental isotopic labeling of HIV-1 capsid protein assemblies for solid state NMR
Abstract
Recent studies of noncrystalline HIV-1 capsid protein (CA) assemblies by our laboratory and by Polenova and coworkers (Protein Sci 19:716â??730, 2010; J Mol Biol 426:1109â??1127, 2014; J Biol Chem 291:13098â??13112, 2016; J Am Chem Soc 138:8538â??8546, 2016; J Am Chem Soc 138:12029â??12032, 2016; J Am Chem Soc 134:6455â??6466, 2012; J Am Chem Soc 132:1976â??1987, 2010; J Am Chem Soc 135:17793â??17803, 2013; Proc Natl Acad Sci USA 112:14617â??14622, 2015; J Am...
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02-21-2018 12:45 AM
[NMR paper] Recent applications of isotopic labeling for protein NMR in drug discovery.
Recent applications of isotopic labeling for protein NMR in drug discovery.
Related Articles Recent applications of isotopic labeling for protein NMR in drug discovery.
Expert Opin Drug Discov. 2013 Mar 12;
Authors: Hiroaki H
Abstract
Introduction: Nuclear magnetic resonance (NMR) applications in drug discovery are classified into two categories: ligand-based methods and protein-based methods. The latter is based on the observation of the (1)H-(15)N HSQC spectra of a protein with and without lead compounds. However, in order to take this...
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03-14-2013 10:05 PM
Segmental isotopic labeling of a 140 kDa dimeric multi-domain protein CheA from Escherichia coli by expressed protein ligation and protein trans-splicing
Segmental isotopic labeling of a 140 kDa dimeric multi-domain protein CheA from Escherichia coli by expressed protein ligation and protein trans-splicing
Abstract Segmental isotopic labeling is a powerful labeling tool to facilitate NMR studies of larger proteins by not only alleviating the signal overlap problem but also retaining features of uniform isotopic labeling. Although two approaches, expressed protein ligation (EPL) and protein trans-splicing (PTS), have been mainly used for segmental isotopic labeling, there has been no single example in which both approaches have been...
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07-02-2012 06:18 AM
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] 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] 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...
Segmental isotopic labeling by asparaginyl endopeptidase-mediated protein ligation
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