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

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• 作者：Scott A. Robson ; Robert Peterson ; Louis-S Bouchard ; Valerie A. Villareal ; Robert T. Clubb
• 刊名：Journal of the American Chemical Society
• 出版年：2010
• 出版时间：July 21, 2010
• 年：2010
• 卷：132
• 期：28
• 页码：9522-9523
• 全文大小：161K
• 年卷期：v.132,no.28(July 21, 2010)
• ISSN：1520-5126

文摘

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 N_z-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 N_z-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 k_off 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 N_z-exchange experiment will expand the usefulness of exchange spectroscopy to slow chemical exchange events that involve ligand binding.