Hustedt E J, Smirnov A I, Laub C F, Cobb C E, Beth A H
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232, USA.
Biophys J. 1997 Apr;72(4):1861-77. doi: 10.1016/S0006-3495(97)78832-5.
For immobilized nitroxide spin-labels with a well-defined interprobe geometry, resolved dipolar splittings can be observed in continuous wave electron paramagnetic resonance (CW-EPR) spectra for interelectron distances as large as 30 A using perdeuterated probes. In this work, algorithms are developed for calculating CW-EPR spectra of immobilized, dipolar coupled nitroxides, and then used to define the limits of sensitivity to the interelectron distance as a function of geometry and microwave frequency. Secondly, the CW-EPR spectra of N epsilon-spin-labeled coenzyme NAD+ bound to microcrystalline, tetrameric glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been collected at 9.8, 34, and 94 GHz. These data have been analyzed, using a combination of simulated annealing and global analysis, to obtain a unique fit to the data. The values of the intermitroxide distance and the five angles defining the relative orientation of the two nitroxides are in reasonable agreement with a molecular model built from the known crystal structure. Finally, the effect of rigid body isotropic rotational diffusion on the CW-EPR spectra of dipolar coupled nitroxides has been investigated using an algorithm based on Brownian dynamics trajectories. These calculations demonstrate the sensitivity of CW-EPR spectra to dipolar coupling in the presence of rigid body rotational diffusion.
对于具有明确探针间几何结构的固定化氮氧自由基自旋标记物，使用全氘化探针时，在连续波电子顺磁共振（CW-EPR）光谱中，对于电子间距高达30 Å的情况，可以观察到分辨的偶极分裂。在这项工作中，开发了用于计算固定化、偶极耦合氮氧自由基的CW-EPR光谱的算法，然后用于确定作为几何结构和微波频率函数的对电子间距的灵敏度极限。其次，在9.8、34和94 GHz下收集了与微晶四聚体甘油醛-3-磷酸脱氢酶（GAPDH）结合的Nε-自旋标记辅酶NAD⁺的CW-EPR光谱。使用模拟退火和全局分析相结合的方法对这些数据进行了分析，以获得对数据的唯一拟合。两个氮氧自由基之间的间距值以及定义两个氮氧自由基相对取向的五个角度与根据已知晶体结构构建的分子模型合理一致。最后，使用基于布朗动力学轨迹的算法研究了刚体各向同性旋转扩散对偶极耦合氮氧自由基的CW-EPR光谱的影响。这些计算证明了在存在刚体旋转扩散的情况下，CW-EPR光谱对偶极耦合的灵敏度。
