¹⁹ F electron-nuclear double resonance (ENDOR) spectroscopy is emerging as a method of choice to determine molecular distances in biomolecules in the angstrom to nanometer range. However, line broadening mechanisms in ¹⁹F ENDOR spectra can obscure the detected spin-dipolar coupling that encodes the distance information, thus limiting the resolution and accessible distance range. So far, the origin of these mechanisms has not been understood. Here, we employ a combined approach of rational molecular design, frequency and time domain ENDOR methods as well as quantum mechanical spin dynamics simulations to analyze these mechanisms. We present the first application of Fourier transform ENDOR to remove power broadening and measure T_2n of the ¹⁹F nucleus. We identify nuclear dipolar couplings between the fluorine and protons up to 14 kHz as a major source of spectral broadening. When removing these interactions by H/D exchange, an unprecedented spectral width of 9 kHz was observed suggesting that, generally, the accessible distance range can be extended. In a spin labeled RNA duplex we were able to predict the spectral ENDOR line width, which in turn enabled us to extract a distance distribution. This study represents a first step towards a quantitative determination of distance distributions in biomolecules from ¹⁹F ENDOR.

中文翻译：

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频域和时域 19F ENDOR 光谱：核偶极耦合在确定距离分布中的作用


¹⁹ F 电子-核双共振 （ENDOR） 光谱正在成为确定生物分子中埃到纳米范围内分子距离的首选方法。然而，¹⁹F ENDOR 光谱中的谱线展宽机制会掩盖检测到的编码距离信息的自旋-偶极耦合，从而限制分辨率和可访问的距离范围。到目前为止，这些机制的起源尚不清楚。在这里，我们采用合理的分子设计、频域和时域 ENDOR 方法以及量子力学自旋动力学模拟的组合方法来分析这些机制。我们提出了傅里叶变换 ENDOR 的第一个应用，以消除幂展宽并测量 ¹⁹F 原子核的 T_2n。我们确定氟和质子之间高达 14 kHz 的核偶极耦合是光谱展宽的主要来源。当通过 H/D 交换消除这些相互作用时，观察到前所未有的 9 kHz 频谱宽度，这表明通常可以扩展可访问的距离范围。在自旋标记的 RNA 双链体中，我们能够预测光谱 ENDOR 线宽，这反过来又使我们能够提取距离分布。这项研究代表了定量测定 ¹⁹F ENDOR 生物分子中距离分布的第一步。