Abstract A straightforward approach for the production of highly deuterated proteins labeled with 13C and 1H at Ile-γ2 methyl positions is described. The utility of the methodology is illustrated with an application involving the half proteasome (360 kDa). High quality 2D Ile 13Cγ2,1Hγ2 HMQC data sets, exploiting the methyl-TROSY principle, are recorded with excellent sensitivity and resolution, that compare favorably with Ile 13Cδ1,1Hδ1 spectra. This labeling scheme adds to a growing list of different approaches that are significantly impacting the utility of solution NMR spectroscopy in studies of supra-molecular systems.
Content Type Journal Article
DOI 10.1007/s10858-010-9449-1
Authors
Amy M. Ruschak, Departments of Molecular Genetics, Biochemistry and Chemistry, The University of Toronto, Toronto, ON M5S 1A8, Canada
Algirdas Velyvis, Departments of Molecular Genetics, Biochemistry and Chemistry, The University of Toronto, Toronto, ON M5S 1A8, Canada
Lewis E. Kay, Departments of Molecular Genetics, Biochemistry and Chemistry, The University of Toronto, Toronto, ON M5S 1A8, Canada
An optimized isotopic labelling strategy of isoleucine-?(2) methyl groups for solution NMR studies of high molecular weight proteins.
An optimized isotopic labelling strategy of isoleucine-?(2) methyl groups for solution NMR studies of high molecular weight proteins.
An optimized isotopic labelling strategy of isoleucine-?(2) methyl groups for solution NMR studies of high molecular weight proteins.
Chem Commun (Camb). 2011 Jul 26;
Authors: Ayala I, Hamelin O, Amero C, Pessey O, Plevin MJ, Gans P, Boisbouvier J
An efficient synthetic route is proposed to produce 2-hydroxy-2-ethyl-3-oxobutanoate for the specific labelling of Ile methyl-?(2) groups in proteins. The (2)H,...
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A simple biosynthetic method for stereospecific resonance assignment of prochiral methyl groups in proteins
A simple biosynthetic method for stereospecific resonance assignment of prochiral methyl groups in proteins
Abstract A new method for stereospecific assignment of prochiral methyl groups in proteins is presented in which protein samples are produced using U-glucose and subsaturating amounts of 2-methyl-acetolactate. The resulting non-uniform labeling pattern allows proR and proS methyl groups to be easily distinguished by their different phases in a constant-time two-dimensional 1H-13C correlation spectra. Protein samples are conveniently prepared using the same media composition as the...
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[NMR paper] Differential isotype labeling strategy for determining the structure of myristoylated
Differential isotype labeling strategy for determining the structure of myristoylated recoverin by NMR spectroscopy.
Related Articles Differential isotype labeling strategy for determining the structure of myristoylated recoverin by NMR spectroscopy.
J Biomol NMR. 1998 Feb;11(2):135-52
Authors: Tanaka T, Ames JB, Kainosho M, Stryer L, Ikura M
The three-dimensional solution structure of recombinant bovine myristoylated recoverin in the Ca(2+)-free state has been refined using an array of isotope-assisted multidimensional heteronuclear NMR...
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11-17-2010 11:06 PM
Strategy for complete NMR assignment of disordered proteins with highly repetitive se
Strategy for complete NMR assignment of disordered proteins with highly repetitive sequences based on resolution-enhanced 5D experiments
Abstract A strategy for complete backbone and side-chain resonance assignment of disordered proteins with highly repetitive sequence is presented. The protocol is based on three resolution-enhanced NMR experiments: 5D HN(CA)CONH provides sequential connectivity, 5D HabCabCONH is utilized to identify amino acid types, and 5D HC(CC-TOCSY)CONH is used to assign the side-chain resonances. The improved resolution was achieved by a combination of high...
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10-06-2010 02:16 AM
Strategy for complete NMR assignment of disordered proteins with highly repetitive se
Strategy for complete NMR assignment of disordered proteins with highly repetitive sequences based on resolution-enhanced 5D experiments.
Strategy for complete NMR assignment of disordered proteins with highly repetitive sequences based on resolution-enhanced 5D experiments.
J Biomol NMR. 2010 Oct 2;
Authors: MotáÄ?ková V, NováÄ?ek J, Zawadzka-Kazimierczuk A, Kazimierczuk K, ZÃ*dek L, Sanderová H, Krásný L, KoźmiÅ?ski W, SklenáÅ? V
A strategy for complete backbone and side-chain resonance assignment of disordered proteins with highly...
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[NMR paper] Global folds of highly deuterated, methyl-protonated proteins by multidimensional NMR
Global folds of highly deuterated, methyl-protonated proteins by multidimensional NMR.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--pubs.acs.org-images-acspubs.jpg Related Articles Global folds of highly deuterated, methyl-protonated proteins by multidimensional NMR.
Biochemistry. 1997 Feb 11;36(6):1389-401
Authors: Gardner KH, Rosen MK, Kay LE
The development of 15N, 13C, 2H multidimensional NMR spectroscopy has facilitated the assignment of backbone and side chain resonances of proteins and protein complexes with molecular masses...
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08-22-2010 03:31 PM
[NMR paper] Global folds of highly deuterated, methyl-protonated proteins by multidimensional NMR
Global folds of highly deuterated, methyl-protonated proteins by multidimensional NMR.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--pubs.acs.org-images-acspubs.jpg Related Articles Global folds of highly deuterated, methyl-protonated proteins by multidimensional NMR.
Biochemistry. 1997 Feb 11;36(6):1389-401
Authors: Gardner KH, Rosen MK, Kay LE
The development of 15N, 13C, 2H multidimensional NMR spectroscopy has facilitated the assignment of backbone and side chain resonances of proteins and protein complexes with molecular masses...
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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...
A simple strategy for 13C,1H labeling at the Ile-γ2 methyl position in highly deuter
Linear Mode
