Abstract
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 "hotspots" 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 ?-helical hairpin.
PMID: 29948439 [PubMed - as supplied by publisher]
Segmental isotope labelling and solid-state NMR of a 12â??Ã?â??59Â*kDa motor protein: identification of structural variability
Segmental isotope labelling and solid-state NMR of a 12â??Ã?â??59Â*kDa motor protein: identification of structural variability
Abstract
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...
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06-12-2018 08:40 AM
[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...
Segmental isotope labelling and solid-state NMR of a 12 × 59*kDa motor protein: identification of structural variability.
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