Mapping backbone dynamics in solution with site-directed spin labeling: GCN4-58 bZip free and bound to DNA
Mapping backbone dynamics in solution with site-directed spin labeling: GCN4-58 bZip free and bound to DNA
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DOI：
10.1021/bi0497906
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发表时间：
2004-06-15
期刊：
影响因子：
2.9
通讯作者：
Hubbell, WL
中科院分区：
文献类型：
--
作者：
Columbus, L;Hubbell, WL
在定点自旋标记中，一个含氮氧基侧链被引入到蛋白质中选定的位置。标记蛋白质的EPR谱编码了有关氮氧化物在纳秒时间尺度上运动的信息，这对蛋白质的旋转扩散、侧链的内部运动和主链的波动都有贡献。在非相互作用(表面)侧链运动的最简单模型中，内部运动的贡献与序列无关，蛋白质旋转扩散的贡献也是如此。因此，骨架运动的差异应该通过比较氮氧化合物在结构上同源位置的序列依赖运动来揭示。为了检验这一模型，沿着Gcn4-58bZip序列引入了氮氧化物侧链，一次一个，核磁共振N-15弛豫实验发现沿着DNA结合区的主干迁移率有一个显著的梯度[Bracken等人]。(1999)J.Mol.比奥尔。285,2133]。利用光谱模拟技术和简单的线宽测量从EPR谱中提取动力学参数，结果显示了与核磁共振弛豫相似的迁移率梯度，表明侧链运动反映了主链运动。此外，还分析了DNA/蛋白质复合体中与序列相关的侧链动力学，这是以前从未用核磁共振松弛方法研究过的。正如预期的那样，骨架运动在DNA结合状态下受到抑制，尽管运动的梯度持续存在，DNA结合部位的残基是最有序的，类似于球状蛋白质上的螺旋。
In site-directed spin labeling, a nitroxide-containing side chain is introduced at selected sites in a protein. The EPR spectrum of the labeled protein encodes information about the motion of the nitroxide on the nanosecond time scale, which has contributions from the rotary diffusion of the protein, from internal motions in the side chain, and from backbone fluctuations. In the simplest model for the motion of noninteracting (surface) side chains, the contribution from the internal motion is sequence independent, as is that from protein rotary diffusion. Hence, differences in backbone motions should be revealed by comparing the sequence-dependent motions of nitroxides at structurally homologous sites. To examine this model, nitroxide side chains were introduced, one at a time, along the GCN4-58 bZip sequence, for which NMR N-15 relaxation experiments have identified a striking gradient of backbone mobility along the DNA-binding region [Bracken et al. (1999) J. Mol. Biol. 285, 2133]. Spectral simulation techniques and a simple line width measure were used to extract dynamical parameters from the EPR spectra, and the results reveal a mobility gradient similar to that observed in NMR relaxation, indicating that side chain motions mirror backbone motions. In addition, the sequence-dependent side chain dynamics were analyzed in the DNA/protein complex, which has not been previously investigated by NMR relaxation methods. As anticipated, the backbone motions are damped in the DNA-bound state, although a gradient of motion persists with residues at the DNA-binding site being the most highly ordered, similar to those of helices on globular proteins.