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A Heteronuclear Zero Quantum Coherence Nz-Exchange Experiment That Resolves Resonance Overlap and Its Application To Measure the Rates of Heme Binding to the IsdC Protein

机译:解决共振重叠的异核零量子相干Nz交换实验及其在测量血红素与IsdC蛋白结合率上的应用

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摘要

Chemical exchange phenomena in NMR spectra can be quantitatively interpreted to measure the rates of ligand binding, as well as conformational and chemical rearrangements. In macromolecules, processes that occur slowly on the chemical shift time scale are frequently studied using 2D heteronuclear ZZ or Nz-exchange spectroscopy. However, to successfully apply this method, peaks arising from each exchanging species must have unique chemical shifts in both dimensions, a condition that is often not satisfied in protein−ligand binding equilibria for 15N nuclei. To overcome the problem of 15N chemical shift degeneracy we developed a heteronuclear zero-quantum (and double-quantum) coherence Nz-exchange experiment that resolves 15N chemical shift degeneracy in the indirect dimension. We demonstrate the utility of this new experiment by measuring the heme binding kinetics of the IsdC protein from Staphylococcus aureus. Because of peak overlap, we could not reliably analyze binding kinetics using conventional methods. However, our new experiment resulted in six well-resolved systems that yielded interpretable data. We measured a relatively slow koff rate of heme from IsdC (<10 s−1), which we interpret as necessary so heme loaded IsdC has time to encounter downstream binding partners to which it passes the heme. The utility of using this new exchange experiment can be easily expanded to 13C nuclei. We expect our heteronuclear zero-quantum coherence Nz-exchange experiment will expand the usefulness of exchange spectroscopy to slow chemical exchange events that involve ligand binding.
机译:可以定量解释NMR光谱中的化学交换现象,以测量配体结合率以及构象和化学重排。在大分子中,经常使用二维异核ZZ或N z 交换光谱研究在化学位移时间尺度上缓慢发生的过程。但是,要成功应用此方法,每个交换物种产生的峰在两个方向上都必须具有独特的化学位移,而 15 N核的蛋白质-配体结合平衡通常不满足这种条件。为克服 15 N化学位移简并性的问题,我们开发了一种异核零量子(和双量子)相干N z 交换实验,用于解决 15 < / sup>在间接维度上的N化学位移简并性。我们通过测量来自金黄色葡萄球菌的IsdC蛋白的血红素结合动力学来证明该新实验的实用性。由于峰重叠,我们无法使用常规方法可靠地分析结合动力学。但是,我们的新实验产生了六个可很好解析的系统,可产生可解释的数据。我们测量了IsdC相对较低的血红素k off 速率(<10 s -1 ),我们认为这是必要的,因此装载血红素的IsdC有时间遇到下游结合伴侣它通过血红素到达。使用此新交换实验的实用程序可以轻松扩展到 13 C核。我们期望我们的异核零量子相干N z -交换实验将扩大交换光谱的作用,以减慢涉及配体结合的化学交换事件。

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  • 来源
    《Journal of the American Chemical Society》 |2010年第28期|p.9522-9523|共2页
  • 作者单位

    University of California, Los Angeles, Department of Chemistry and Biochemistry, UCLA-DOE Institute for Genomics and Proteomics and the California NanoSystems Institute, UCLA, 611 Charles E. Young Drive, Los Angeles, California 90095;

  • 收录信息 美国《科学引文索引》(SCI);美国《工程索引》(EI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
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  • 入库时间 2022-08-18 00:50:17

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