Segmental isotope labelling enables the NMR study of an individual domain within a multidomain protein, but still in the context of the entire full-length protein. Compared to the fully labelled protein, spectral overlap can be greatly reduced. We here describe segmental labelling of the (double-) hexameric DnaB helicase from Helicobacter pylori using a ligation approach. Solid-state spectra demonstrate that the ligated protein has the same structure and structural order as the directly expressed full-length protein. We uniformly 13C/15N labeled the N-terminal domain (147 residues) of the protein, while the C-terminal domain (311 residues) remained in natural abundance. The reduced signal overlap in solid-state NMR spectra allowed to identify structural â??hotspotsâ?? for which the structure of the N-terminal domain in the context of the oligomeric full-length protein differs from the one in the isolated form. They are located near the linker between the two domains, in an α-helical hairpin.
[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] Protein-nucleotide contacts in motor proteins detected by DNP-enhanced solid-state NMR.
Protein-nucleotide contacts in motor proteins detected by DNP-enhanced solid-state NMR.
Related Articles Protein-nucleotide contacts in motor proteins detected by DNP-enhanced solid-state NMR.
J Biomol NMR. 2017 Nov 08;:
Authors: Wiegand T, Liao WC, Ong TC, Däpp A, Cadalbert R, Copéret C, Böckmann A, Meier BH
Abstract
DNP (dynamic nuclear polarization)-enhanced solid-state NMR is employed to directly detect protein-DNA and protein-ATP interactions and identify the residue type establishing the intermolecular contacts. While...
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11-10-2017 05:01 PM
Proteinâ??nucleotide contacts in motor proteins detected by DNP-enhanced solid-state NMR
Proteinâ??nucleotide contacts in motor proteins detected by DNP-enhanced solid-state NMR
Abstract
DNP (dynamic nuclear polarization)-enhanced solid-state NMR is employed to directly detect proteinâ??DNA and proteinâ??ATP interactions and identify the residue type establishing the intermolecular contacts. While conventional solid-state NMR can detect proteinâ??DNA interactions in large oligomeric protein assemblies in favorable cases, it typically suffers from low signal-to-noise ratios. We show here, for the oligomeric DnaB helicase from...
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11-09-2017 08:55 AM
[NMR paper] Unambiguous Assignment of Short- and Long-Range Structural Restraints by Solid-State NMR Spectroscopy with Segmental Isotope Labeling.
Unambiguous Assignment of Short- and Long-Range Structural Restraints by Solid-State NMR Spectroscopy with Segmental Isotope Labeling.
Related Articles Unambiguous Assignment of Short- and Long-Range Structural Restraints by Solid-State NMR Spectroscopy with Segmental Isotope Labeling.
Chembiochem. 2014 Nov 12;
Authors: Schubeis T, Lührs T, Ritter C
Abstract
We present an efficient method for the reduction of spectral complexity in the solid-state NMR spectra of insoluble protein assemblies, without loss of signal intensity....
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11-14-2014 08:40 PM
[NMR paper] Amino Acid-Selective Segmental Isotope Labeling of Multidomain Proteins for Structural Biology.
Amino Acid-Selective Segmental Isotope Labeling of Multidomain Proteins for Structural Biology.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--media.wiley.com-assets-2250-98-WileyOnlineLibrary-Button_120x27px_FullText.gif Related Articles Amino Acid-Selective Segmental Isotope Labeling of Multidomain Proteins for Structural Biology.
Chembiochem. 2013 Jan 30;
Authors: Michel E, Skrisovska L, Wüthrich K, Allain FH
Abstract
Current solution NMR techniques enable structural investigations of proteins in molecular particles with sizes...
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02-03-2013 10:19 AM
Segmental isotope labeling of proteins for NMR structural study using a protein S tag for higher expression and solubility
Segmental isotope labeling of proteins for NMR structural study using a protein S tag for higher expression and solubility
Abstract A common obstacle to NMR studies of proteins is sample preparation. In many cases, proteins targeted for NMR studies are poorly expressed and/or expressed in insoluble forms. Here, we describe a novel approach to overcome these problems. In the protein S tag-intein (PSTI) technology, two tandem 92-residue N-terminal domains of protein S (PrS2) from Myxococcus xanthus is fused at the N-terminal end of a protein to enhance its expression and solubility. Using...
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03-08-2012 08:46 AM
Suppression of isotope scrambling in cell-free protein synthesis by broadband inhibition of PLP enymes for selective 15N-labelling and production of perdeuterated proteins in H2O
Suppression of isotope scrambling in cell-free protein synthesis by broadband inhibition of PLP enymes for selective 15N-labelling and production of perdeuterated proteins in H2O
Abstract Selectively isotope labelled protein samples can be prepared in vivo or in vitro from selectively labelled amino acids but, in many cases, metabolic conversions between different amino acids result in isotope scrambling. The best results are obtained by cell-free protein synthesis, where metabolic enzymes are generally less active, but isotope scrambling can never be suppressed completely. We show that...
Segmental isotope labelling and solid-state NMR of a 12â??Ã?â??59Â*kDa motor protein: identification of structural variability
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