Related ArticlesBiosynthetic site-specific (13) C labeling of the light-harvesting 2 protein complex: a model for solid state NMR structure determination of transmembrane proteins.
J Biomol NMR. 2004 Nov;30(3):267-74
Authors: van Gammeren AJ, Hulsbergen FB, Hollander JG, de Groot HJ
Partly biosynthetic site-directed isotopically (13)C enriched photosynthetic light-harvesting 2(LH2) complexes have been prepared from Rhodopseudomonas acidophila strain 10050 by using chemically labeled [1,2,3,4-(13)C], [1,4-(13)C] and [2,3-(13)C] succinic acid as a precursor in the growth medium. Two-dimensional proton driven spin diffusion (PDSD) solid state NMR correlation spectroscopy has been used to trace each individual (13)C isotope from the labeled succinic acid precursor to its destination into the protein and into the embedded major light-absorbing bacteriochlorophyll cofactors. For both the residues of the protein and for the cofactors distinct labeling patterns have been deduced, for protein complexes prepared from [1,4-(13)C]-succinic acid or [2,3-(13)C]-succinic labeled media. All residues, except isoleucine and leucine, have been labeled almost homogeneously by the succinic acid precursor. Carbonyl carbons in the protein backbone were labeled by [1,4-(13)C]-succinic acid, while the Calpha and Cbeta carbons of the residues were labeled by [2,3 (13)C]-succinic acid. Leucine and isoleucine residues were labeled using a uniformly labeled amino acid mixture in the medium. The pattern labeling yields an increase of the resolution and less spectral crowding. The partial labeling technique in combination with conventional solid state NMR methods at ultra high magnetic fields provides an attractive route to resolve chemical shifts for alpha-helical transmembrane protein structures.
Solid-state NMR applied to photosynthetic light-harvesting complexes.
Solid-state NMR applied to photosynthetic light-harvesting complexes.
Solid-state NMR applied to photosynthetic light-harvesting complexes.
Photosynth Res. 2011 Aug 13;
Authors: Pandit A, de Groot HJ
This short review describes how solid-state NMR has provided a mechanistic and electronic picture of pigment-protein and pigment-pigment interactions in photosynthetic antenna complexes. NMR results on purple bacterial antenna complexes show how the packing of the protein and the pigments inside the light-harvesting oligomers induces mutual...
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08-16-2011 01:19 PM
First solid-state NMR analysis of uniformly (13)C-enriched major light-harvesting complexes from Chlamydomonas reinhardtti and identification of protein and cofactor spin clusters.
First solid-state NMR analysis of uniformly (13)C-enriched major light-harvesting complexes from Chlamydomonas reinhardtti and identification of protein and cofactor spin clusters.
First solid-state NMR analysis of uniformly (13)C-enriched major light-harvesting complexes from Chlamydomonas reinhardtti and identification of protein and cofactor spin clusters.
Biochim Biophys Acta. 2011 Jan 25;
Authors: Pandit A, Morosinotto T, Reus M, Holzwarth AR, Bassi R, de Groot HJ
The light-harvesting complex II (LHCII) is the main component of the...
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02-02-2011 02:40 AM
Site-specific labeling of proteins with NMR-active unnatural amino acids
Site-specific labeling of proteins with NMR-active unnatural amino acids
Abstract A large number of amino acids other than the canonical amino acids can now be easily incorporated in vivo into proteins at genetically encoded positions. The technology requires an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is added to the media while a TAG amber or frame shift codon specifies the incorporation site in the protein to be studied. These unnatural amino acids can be isotopically labeled and provide unique opportunities for site-specific labeling...
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01-09-2011 12:46 PM
Optimizing 19F NMR protein spectroscopy by fractional biosynthetic labeling
Abstract In protein NMR experiments which employ nonnative labeling, incomplete enrichment is often associated with inhomogeneous line broadening due to the presence of multiple labeled species. We investigate the merits of fractional enrichment strategies using a monofluorinated phenylalanine species, where resolution is dramatically improved over that achieved by complete enrichment. In NMR studies of calmodulin, a 148 residue calcium binding protein, 19F and 1H-15N HSQC spectra reveal a significant extent of line broadening and the appearance of minor conformers in the presence of...
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08-25-2010 03:51 PM
Optimizing (19)F NMR protein spectroscopy by fractional biosynthetic labeling.
Optimizing (19)F NMR protein spectroscopy by fractional biosynthetic labeling.
Related Articles Optimizing (19)F NMR protein spectroscopy by fractional biosynthetic labeling.
J Biomol NMR. 2010 Aug 24;
Authors: Kitevski-Leblanc JL, Evanics F, Scott Prosser R
In protein NMR experiments which employ nonnative labeling, incomplete enrichment is often associated with inhomogeneous line broadening due to the presence of multiple labeled species. We investigate the merits of fractional enrichment strategies using a monofluorinated phenylalanine species,...
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08-25-2010 02:04 PM
[NMR paper] Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-N
Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-NMR titration of Trp 62-lysozyme.
Related Articles Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-NMR titration of Trp 62-lysozyme.
J Biochem. 1991 Aug;110(2):295-300
Authors: Nakazawa T, Sakiyama F
The indole C-2(delta 1) carbon of Trp 62 in hen egg-white lysozyme was selectively labeled with 13C through a series of reactions involving N'-formylkynurenine 62-lysozyme with K13CN, NaBH4-reduction, and acid-catalyzed...
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08-21-2010 11:12 PM
[NMR paper] Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-N
Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-NMR titration of Trp 62-lysozyme.
Related Articles Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-NMR titration of Trp 62-lysozyme.
J Biochem. 1991 Aug;110(2):295-300
Authors: Nakazawa T, Sakiyama F
The indole C-2(delta 1) carbon of Trp 62 in hen egg-white lysozyme was selectively labeled with 13C through a series of reactions involving N'-formylkynurenine 62-lysozyme with K13CN, NaBH4-reduction, and acid-catalyzed...
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08-21-2010 11:12 PM
Site-specific labeling of nucleotides for making RNA for high resolution NMR studies
Abstract Escherichia coli (E. coli) is a versatile organism for making nucleotides labeled with stable isotopes (13C, 15N, and/or 2H) for structural and molecular dynamics characterizations. Growth of a mutant E. coli strain deficient in the pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase (K10-1516) on 2-13C-glycerol and 15N-ammonium sulfate in Studier minimal medium enables labeling at sites useful for NMR spectroscopy. However, 13C-sodium formate combined with 13C-2-glycerol in the growth media adds labels to new positions. In the absence of labeled formate, both C5 and...
Biosynthetic site-specific (13) C labeling of the light-harvesting 2 protein complex:
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