Department of Chemistry and Biochemistry, Jules Stein Eye Institute, University of California, Los Angeles, CA 90095-7008, USA.
Biochemistry. 2012 Aug 21;51(33):6568-83. doi: 10.1021/bi3005686. Epub 2012 Aug 9.
Site-directed spin labeling (SDSL) has potential for mapping protein flexibility under physiological conditions. The purpose of the present study was to explore this potential using 38 singly spin-labeled mutants of myoglobin distributed throughout the sequence. Correlation of the EPR spectra with protein structure provides new evidence that the site-dependent variation in line shape, and hence motion of the spin label, is due largely to differences in mobility of the helical backbone in the ns time range. Fluctuations between conformational substates, typically in the μs-ms time range, are slow on the EPR time scale, and the spectra provide a snapshot of conformational equilibria frozen in time as revealed by multiple components in the spectra. A recent study showed that osmolyte perturbation can positively identify conformational exchange as the origin of multicomponent spectra (López et al. (2009), Protein Sci. 18, 1637). In the present study, this new strategy is employed in combination with line shape analysis and pulsed-EPR interspin distance measurements to investigate the conformation and flexibility of myoglobin in three folded and partially folded states. The regions identified to be in conformational exchange in the three forms agree remarkably well with those assigned by NMR, but the faster time scale of EPR allows characterization of localized states not detected in NMR. Collectively, the results suggest that SDSL-EPR and osmolyte perturbation provide a facile means for mapping the amplitude of fast backbone fluctuations and for detecting sequences in slow conformational exchange in folded and partially folded protein sequences.
定点自旋标记（SDSL）在生理条件下绘制蛋白质柔韧性图谱具有很大的潜力。本研究的目的是利用遍布肌红蛋白序列的 38 种单一自旋标记突变体来探索这种潜力。EPR 光谱与蛋白质结构的相关性提供了新的证据，表明谱线形状的位置依赖性变化，因此自旋标记的运动，主要是由于螺旋骨架在 ns 时间范围内的流动性差异。构象亚稳态之间的波动，通常在 μs-ms 时间范围内，在 EPR 时间尺度上非常缓慢，并且光谱提供了时间冻结的构象平衡的快照，这是由光谱中的多个分量揭示的。最近的一项研究表明，渗透压剂的干扰可以积极识别构象交换，作为多分量光谱的来源（López 等人，2009 年，《蛋白质科学》18,1637）。在本研究中，这种新策略与谱线形状分析和脉冲 EPR 自旋间距离测量相结合，用于研究三种折叠和部分折叠状态下肌红蛋白的构象和柔韧性。在三种形式中确定处于构象交换的区域与通过 NMR 分配的区域非常吻合，但 EPR 的更快时间尺度允许对 NMR 中未检测到的局部状态进行特征描述。总的来说，结果表明 SDSL-EPR 和渗透压剂干扰为绘制快速骨架波动幅度提供了一种简单的方法，并可用于检测折叠和部分折叠蛋白质序列中缓慢构象交换的序列。
