Chain-selective isotopic labeling for NMR studies of large multimeric proteins: application to hemoglobin.
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.
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 USA.
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Simplaceanu
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Lukin
T Y
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Fang
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Zou
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Ho
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Ho
D001665:Binding Sites; D002263:Carboxyhemoglobin / Q000737:chemistry*; D003001:Cloning, Molecular; D004926:Escherichia coli; D005813:Genes, Synthetic; D006418:Heme / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics; D006639:Histidine; D006801:Humans; D007553:Isotope Labeling; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry
D006454:Hemoglobins; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D006418:Heme; D006639:Histidine; D002263:Carboxyhemoglobin