Department of Chemistry and Chemical Biology, and ACERT, National Biomedical Center for Advanced Electron Spin Resonance Technology, Cornell University, Ithaca, New York 14853-1301, United States.
In Vivo Multifunctional Magnetic Resonance Center, Robert C. Byrd Health Sciences Center, West Virginia University, and Department of Biochemistry, West Virginia University School of Medicine, Morgantown, West Virginia 26506, United States.
J Am Chem Soc. 2020 Dec 23;142(51):21368-21381. doi: 10.1021/jacs.0c09469. Epub 2020 Dec 11.
Exchange processes which include conformational change, protonation/deprotonation, and binding equilibria are routinely studied by 2D exchange NMR techniques, where information about the exchange of nuclei between environments with different NMR shifts is obtained from the development of cross-peaks. Whereas 2D NMR enables the real time study of millisecond and slower exchange processes, 2D ESR in the form of 2D-ELDOR (two-dimensional electron-electron double resonance) has the potential for such studies over the nanosecond to microsecond real time scales. Cross-peak development due to chemical exchange has been seen previously for semiquinones in ESR, but this is not possible for most common ESR probes, such as nitroxides, studied at typical ESR frequencies because, unlike NMR, the exchanging states yield ESR signals that are not resolved from each other within their respective line widths. But at 95 GHz, it becomes possible to resolve them in many cases because of the increased -factor resolution. The 95 GHz instrumental developments occurring at ACERT now enable such studies. We demonstrate these new capabilities in two studies: (A) the protonation/deprotonation process for a pH-sensitive imidazoline spin label in aqueous solution where the exchange rate and the population ratio of the exchanging states are controlled by the concentration and pH of the buffer solution, respectively, and (B) a nitroxide radical partitioning between polar (aqueous) and nonpolar (phospholipid) environments in multilamellar lipid vesicles, where the cross-peak development arises from the exchange of the nitroxide between the two phases. This work represents the first example of the observation and analysis of cross-peaks arising from chemical exchange processes involving nitroxide spin labels.
交换过程包括构象变化、质子化/去质子化和结合平衡，通常通过二维交换 NMR 技术进行研究，其中通过交叉峰的发展获得关于核在具有不同 NMR 位移的环境之间交换的信息。虽然 2D NMR 能够实时研究毫秒级和更慢的交换过程，但以二维 ELDOR（二维电子-电子双共振）形式存在的 2D ESR 具有在纳秒到微秒实时范围内进行此类研究的潜力。以前在 ESR 中已经看到过半醌类物质的化学交换导致的交叉峰发展，但对于大多数常见的 ESR 探针（如氮氧自由基），这种情况是不可能的，因为与 NMR 不同，交换状态产生的 ESR 信号在各自的线宽内彼此无法分辨。但在 95 GHz 下，由于 -因子分辨率的增加，在许多情况下可以分辨它们。ACERT 目前正在进行的 95 GHz 仪器开发使这种研究成为可能。我们在两项研究中展示了这些新的能力：（A）在水溶液中 pH 敏感的咪唑啉自旋标记的质子化/去质子化过程，其中交换速率和交换状态的种群比分别由缓冲溶液的浓度和 pH 控制，（B）在多层脂质泡囊中的极性（水相）和非极性（磷脂）环境之间的氮氧自由基分相，其中交叉峰的发展源于氮氧自由基在两相之间的交换。这项工作代表了观察和分析涉及氮氧自由基自旋标记的化学交换过程产生的交叉峰的第一个例子。
