Centre for Advanced Electron Spin Resonance and Inorganic Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3QR, UK.
Department of Chemical Sciences, University of Padova, 35131 Padova, Italy.
Molecules. 2022 Nov 3;27(21):7526. doi: 10.3390/molecules27217526.
We present a new photoswitchable spin label for light-induced pulsed electron paramagnetic resonance dipolar spectroscopy (LiPDS), the photoexcited triplet state of erythrosin B (EB), which is ideal for biological applications. With this label, we perform an in-depth study of the orientational effects in dipolar traces acquired using the refocused laser-induced magnetic dipole technique to obtain information on the distance and relative orientation between the EB and nitroxide labels in a rigid model peptide, in good agreement with density functional theory predictions. Additionally, we show that these orientational effects can be averaged to enable an orientation-independent analysis to determine the distance distribution. Furthermore, we demonstrate the feasibility of these experiments above liquid nitrogen temperatures, removing the need for expensive liquid helium or cryogen-free cryostats. The variety of choices in photoswitchable spin labels and the affordability of the experiments are critical for LiPDS to become a widespread methodology in structural biology.
我们提出了一种新的光诱导脉冲电子顺磁共振双共振（LiPDS）光开关自旋标记物，即曙红 B（EB）的光激发三重态，这非常适合生物应用。使用这个标记物，我们使用再聚焦激光诱导磁偶极技术获取的偶极轨迹中的各向异性进行了深入研究，以获得有关刚性模型肽中 EB 和氮氧自由基标记物之间距离和相对取向的信息，与密度泛函理论预测非常吻合。此外，我们表明这些各向异性可以进行平均，从而实现一种不依赖于取向的分析方法来确定距离分布。此外，我们证明了这些实验在液氮温度以上的可行性，无需昂贵的液氦或无液氦低温恒温器。光开关自旋标记物的各种选择以及实验的经济性对于 LiPDS 成为结构生物学中广泛应用的方法至关重要。
