Measurement of rotational molecular motion by time-resolved saturation transfer electron paramagnetic resonance.
Measurement of rotational molecular motion by time-resolved saturation transfer electron paramagnetic resonance.
复制标题
通过时间分辨饱和转移电子顺磁共振测量旋转分子运动。
DOI：
10.1016/s0006-3495(86)83562-7
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发表时间：
1986
影响因子：
3.4
通讯作者：
Hyde,JS
中科院分区：
文献类型：
--
作者：
Fajer,P;Thomas,DD;Feix,JB;Hyde,JS
我们使用饱和恢复电子顺磁共振(SR-EPR)，一种时间分辨的饱和转移EPR技术，直接测量了自旋标记蛋白质的微秒旋转扩散。SR-EPR使用强烈的微波脉冲来饱和相对于磁场具有窄取向分布的自旋布居。然后观察信号的时间演化。随着自旋晶格弛豫(TL)和光谱扩散(TSD)引起的饱和转移的解除，信号随时间增加，这是旋转扩散(Tr)和光谱位置的函数。在这两个事件的存在下，恢复是两相的，初始阶段与Tr和Tl有关，第二阶段仅由Tl决定。我们测量了自旋标记的血红蛋白在已知粘度的介质中滚动的饱和恢复率作为旋转相关时间(Tr)和脉冲持续时间(Tp)的函数。从恢复初期估算的Tr值与理论符合得很好。脉冲时间的变化也可以用来确定Tr。在TP小于TSD的情况下，观察到恢复是双相的，而对于TP大于TSD的情况，则是单指数的。T1值是从脉冲猝灭光谱扩散后的恢复或较短脉冲后恢复的第二阶段确定的。这些结果表明，SR-EPR适用于自旋标记蛋白质的运动研究。它的时间分辨率应该比稳态技术有显著的优势，特别是在运动各向异性或系统非均质性的情况下。
We have used saturation-recovery electron paramagnetic resonance (SR-EPR), a time-resolved saturation transfer EPR technique, to measure directly the microsecond rotational diffusion of spin-labeled proteins. SR-EPR uses an intense microwave pulse to saturate a spin population having narrow distribution of orientations with respect to the magnetic field. The time evolution of the signal is then observed. The signal increases in time as saturation is relieved by spin-lattice relaxation (Tl) as well as by saturation transfer due to spectral diffusion (Tsd), which is a function of rotational diffusion (Tr) and spectral position. In the presence of both events, the recovery is biphasic, with the initial phase related to both Tr and Tl, and the second phase determined only by Tl. We have measured the saturation recoveries of spin-labeled hemoglobin tumbling in media of known viscosities as a function of rotational correlation time (Tr) and pulse duration (tp). The Tr values estimated from the initial phase of recovery were in good agreement with theory. Variation of the pulse time can also be used to determine Tr. For tp less than Tsd, the recoveries were observed to be biphasic, for tp greater than Tsd a single-exponential. T1 values were determined from the recoveries after pulses quenching spectral diffusion or from the second phase of recovery after shorter pulses. These results demonstrate that SR-EPR is applicable to the study of motion of spin-labeled proteins. Its time resolution should provide a significant advantage over steady state techniques, particularly in the case of motional anisotropy or system heterogeneity.