Related ArticlesChain-selective isotopic labeling for NMR studies of large multimeric proteins: application to hemoglobin.
Biophys J. 2000 Aug;79(2):1146-54
Authors: Simplaceanu V, Lukin JA, Fang TY, Zou M, Ho NT, Ho C
Multidimensional, multinuclear NMR has the potential to elucidate the mechanisms of allostery and cooperativity in multimeric proteins under near-physiological conditions. However, NMR studies of proteins made up of non-equivalent subunits face the problem of severe resonance overlap, which can prevent the unambiguous assignment of resonances, a necessary step in interpreting the spectra. We report the application of a chain-selective labeling technique, in which one type of subunit is labeled at a time, to carbonmonoxy-hemoglobin A (HbCO A). This labeling method can be used to extend previous resonance assignments of key amino acid residues, which are important to the physiological function of hemoglobin. Among these amino acid residues are the surface histidyls, which account for the majority of the Bohr effect. In the present work, we report the results of two-dimensional heteronuclear multiple quantum coherence (HMQC) experiments performed on recombinant (15)N-labeled HbCO A. In addition to the C2-proton (H epsilon(1)) chemical shifts, these spectra also reveal the corresponding C4-proton (H delta(2)) resonances, correlated with the N epsilon(2) and N delta(1) chemical shifts of all 13 surface histidines per alpha beta dimer. The HMQC spectrum also allows the assignment of the H delta(1), H epsilon(1), and N epsilon(1) resonances of all three tryptophan residues per alpha beta dimer in HbCO A. These results indicate that heteronuclear NMR, used with chain-selective isotopic labeling, can provide resonance assignments of key regions in large, multimeric proteins, suggesting an approach to elucidating the solution structure of hemoglobin, a protein with molecular weight 64.5 kDa.
Frequency-selective heteronuclear dephasing and selective carbonyl labeling to deconvolute crowded spectra of membrane proteins by magic angle spinning NMR.
Frequency-selective heteronuclear dephasing and selective carbonyl labeling to deconvolute crowded spectra of membrane proteins by magic angle spinning NMR.
Frequency-selective heteronuclear dephasing and selective carbonyl labeling to deconvolute crowded spectra of membrane proteins by magic angle spinning NMR.
J Magn Reson. 2011 Mar 17;
Authors: Traaseth NJ, Veglia G
We present a new method that combines carbonyl-selective labeling with frequency-selective heteronuclear recoupling to resolve the spectral overlap of magic angle spinning (MAS) NMR...
nmrlearner
Journal club
0
04-13-2011 11:57 PM
Frequency-Selective Heteronuclear Dephasing and Selective Carbonyl Labeling to Deconvolute Crowded Spectra of Membrane Proteins By Magic Angle Spinning NMR
Frequency-Selective Heteronuclear Dephasing and Selective Carbonyl Labeling to Deconvolute Crowded Spectra of Membrane Proteins By Magic Angle Spinning NMR
Publication year: 2011
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 17 March 2011</br>
Nathaniel J., Traaseth , Gianluigi, Veglia</br>
We present a new method that combines carbonyl-selective labeling with frequency-selective heteronuclear recoupling to resolve the spectral overlap of magic angle spinning (MAS) NMR spectra of membrane proteins in fluid lipid membranes with broad lines and...
nmrlearner
Journal club
0
03-18-2011 06:43 AM
Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle.
Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle.
Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle.
J Biomol NMR. 2010 Dec;48(4):179-92
Authors: Thakur CS, Sama JN, Jackson ME, Chen B, Dayie TK
Escherichia coli (E. coli) is an ideal organism to tailor-make labeled nucleotides for biophysical studies of RNA. Recently, we showed that adding labeled formate enhanced the isotopic enrichment at protonated carbon...
nmrlearner
Journal club
0
03-01-2011 12:14 PM
[NMR paper] Amino acid-specific isotopic labeling and active site NMR studies of iron(II)- and ir
Amino acid-specific isotopic labeling and active site NMR studies of iron(II)- and iron(III)-superoxide dismutase from Escherichia coli.
Related Articles Amino acid-specific isotopic labeling and active site NMR studies of iron(II)- and iron(III)-superoxide dismutase from Escherichia coli.
J Biomol NMR. 2000 Aug;17(4):311-22
Authors: Sorkin DL, Miller AF
We have developed and employed multiple amino acid-specific isotopic labeling schemes to obtain definitive assignments for active site 1H NMR resonances of iron(II)- and iron(III)-superoxide...
nmrlearner
Journal club
0
11-19-2010 08:29 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...
nmrlearner
Journal club
0
11-18-2010 07:05 PM
Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli
Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle
Abstract Escherichia coli (E. coli) is an ideal organism to tailor-make labeled nucleotides for biophysical studies of RNA. Recently, we showed that adding labeled formate enhanced the isotopic enrichment at protonated carbon sites in nucleotides. In this paper, we show that growth of a mutant E. coli strain DL323 (lacking succinate and malate dehydrogenases) on 13C-2-glycerol and 13C-1,3-glycerol enables selective labeling at many useful sites for RNA NMR...
nmrlearner
Journal club
0
11-09-2010 03:17 PM
[NMR paper] Protein expression, selective isotopic labeling, and analysis of hyperfine-shifted NM
Protein expression, selective isotopic labeling, and analysis of hyperfine-shifted NMR signals of Anabaena 7120 vegetative ferredoxin.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--linkinghub.elsevier.com-ihub-images-PubMedLink.gif Related Articles Protein expression, selective isotopic labeling, and analysis of hyperfine-shifted NMR signals of Anabaena 7120 vegetative ferredoxin.
Arch Biochem Biophys. 1995 Jan 10;316(1):619-34
Authors: Cheng H, Westler WM, Xia B, Oh BH, Markley JL
Two alternative T7 RNA promoter/polymerase systems...
nmrlearner
Journal club
0
08-22-2010 03:41 AM
High Resolution 1H Detected 1H,13C Correlation Spectra in MAS Solid-State NMR using Deuterated Proteins with Selective 1H,2H Isotopic Labeling of Methyl Groups
High Resolution <SUP>1</SUP>H Detected <SUP>1</SUP>H,<SUP>13</SUP>C Correlation Spectra in MAS Solid-State NMR using Deuterated Proteins with Selective <SUP>1</SUP>H,<SUP>2</SUP>H Isotopic Labeling of Methyl Groups
Vipin Agarwal, Anne Diehl, Nikolai Skrynnikov, and Bernd Reif
J. Am. Chem. Soc.; 2006; 128(39) pp 12620 - 12621;
Abstract:
MAS solid-state NMR experiments applied to biological solids are still hampered by low sensitivity and resolution. In this work, we employ a deuteration scheme in which individual methyl groups are selectively protonated. This labeling scheme...
Chain-selective isotopic labeling for NMR studies of large multimeric proteins: appli
Linear Mode
