Chain-selective isotopic labeling for NMR studies of large multimeric proteins: Application to hemoglobin
Chain-selective isotopic labeling for NMR studies of large multimeric proteins: Application to hemoglobin
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DOI：
10.1016/s0006-3495(00)76368-5
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发表时间：
2000-08-01
影响因子：
3.4
通讯作者：
Ho, C
中科院分区：
文献类型：
--
作者：
Simplaceanu, V;Lukin, JA;Ho, C
多维、多核核磁共振有潜力阐明近生理条件下多聚体蛋白的变构和协同作用机制。然而，对由非等效亚基组成的蛋白质的核磁共振研究面临严重的共振重叠问题，这可能会阻碍对共振的明确分配，这是解释光谱的必要步骤。我们报告了一种链选择标记技术的应用，其中一种类型的亚基一次被标记为碳氧血红蛋白a （HbCO a）。这种标记方法可用于扩展先前对血红蛋白生理功能重要的关键氨基酸残基的共振分配。在这些氨基酸残基中，表面组氨酸占玻尔效应的大部分。在本工作中，我们报告了在重组N-15标记的HbCO a上进行的二维异核多重量子相干（HMQC）实验的结果。除了c2 -质子（H - epsilon(1)）化学位移外，这些光谱还揭示了相应的c4 -质子（H - delta(2)）共振，这些共振与α - β二聚体中所有13种表面组氨酸的N- epsilon(2)和N- delta(1)化学位移相关。HMQC光谱还允许分配每个α - β二聚体中所有三个色氨酸残基的H delta(1), H epsilon(1)和N epsilon(1)的共振。这些结果表明，异核NMR与链选择性同位素标记一起使用，可以提供大的多聚体蛋白质中关键区域的共振分配，提出了一种阐明血红蛋白溶液结构的方法，血红蛋白分子量为64.5 kDa。
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 N-15-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.