Rotational dynamics of HIV-1 nucleocapsid protein NCp7 as probed by a spin label attached by peptide synthesis.
Rotational dynamics of HIV-1 nucleocapsid protein NCp7 as probed by a spin label attached by peptide synthesis.
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通过肽合成附着的自旋标签探测 HIV-1 核衣壳蛋白 NCp7 的旋转动力学。
DOI：
10.1002/bip.21064
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发表时间：
2008
期刊：
影响因子：
2.9
通讯作者：
Karim,ChristineB
中科院分区：
文献类型：
--
作者：
Zhang,Zhiwen;Xi,Xiangmei;Scholes,CharlesP;Karim,ChristineB
2,2,6,6-四甲基哌啶-1-氧基-4-氨基-4-羧酸 (TOAC) 自旋标签连接在 N 末端位置，以检测 HIV-1 核衣壳锌指蛋白 NCp7 的动力学。 NCp7 是一种 6.4 kDa 55 聚体，对于具有茎环结构的病毒 RNA（例如本研究中使用的 RNA 茎环 3）的识别、包装和高效逆转录至关重要。 NCp7 通过固相肽合成法制备，TOAC 并入 α-碳主链的 N 端“0”位置，显示出分析纯度和生物活性。 N 端 TOAC 的电子顺磁共振 (EPR) 谱表明探针在柔性 N 端段上的快速温度敏感运动（≤0.33 ns 相关时间）。该 N 末端 TOAC-NCp7 报告了 NCp7 与 RNA 比例为 1:1 的 RNA-NCp7 相互作用，这导致翻滚时间从约 0.3 ns 减慢至约 0.5 ns。 NCp7 是一种很大程度上无序的蛋白质，可以适应其 RNA 靶标。然而，如圆二色性所示，≥90%的三氟乙醇[(TFE)，一种α-螺旋增强剂]导致手指中没有锌的TOAC-NCp7变成完全螺旋构象，而TOAC自旋标签同时报告远超过纳秒的翻滚时间，因为N端TOAC变得不灵活地折叠。即使存在 TFE，NCp7 中完整锌指区域的存在也阻止了螺旋结构的完全形成（如圆二色性所示），并减少了 N 末端 TOAC 翻滚时间（如 EPR 所示）。这项研究证明 NCp7 N 末端的 TOAC 是 NCp7 相当大的构象不稳定性的报告基因。 © 2008 Wiley periodicals, Inc. Biopolymers 89: 1125–1135, 2008。本文最初作为已接受的预印本在网上发表。 “在线发布”日期对应于预印本版本。您可以通过发送电子邮件至 Biopolymers 编辑部索取预印本副本：biopolymers@wiley.com
2,2,6,6‐Tetramethylpiperidine‐1‐oxyl‐4‐amino‐4‐carboxylic acid (TOAC) spin label was attached at the N‐terminal position to interrogate the dynamics of the HIV‐1 nucleocapsid Zn‐finger protein, NCp7. NCp7 is a 6.4‐kDa 55‐mer critical to the recognition, packaging, and efficient reverse transcription of viral RNA that has stem‐loop structures, such as the RNA stem‐loop 3 used in this work. The NCp7, made by solid‐phase peptide synthesis with TOAC incorporated into the α‐carbon backbone at the N‐terminal “0” position, showed analytical purity and biological activity. Electron Paramagnetic Resonance (EPR) spectra of the N‐terminal TOAC indicated rapid temperature‐sensitive motion of the probe (≤0.33 ns correlation time) on the flexible N‐terminal segment. This N‐terminal TOAC‐NCp7 reported a RNA‐NCp7 interaction at a 1:1 ratio of NCp7 to RNA, which caused the tumbling time to be slowed from about 0.3 ns to about 0.5 ns. NCp7 is a largely disordered protein that adapts to its RNA targets. However, as shown by circular dichroism, ≥90% trifluoroethanol [(TFE), an α‐helix enhancer] caused the TOAC‐NCp7 without zinc in its fingers to change to a fully helical conformation, while the TOAC spin label was concurrently reporting a tumbling time of well over a nanosecond, as the N‐terminal TOAC became inflexibly enfolded. Even with TFE present, the existence of intact Zn‐finger regions in NCp7 prevented complete formation of helical structure, as shown by circular dichroism, and decreased the N‐terminal TOAC tumbling time, as shown by EPR. This study demonstrated TOAC at the N‐terminal of NCp7 to be a reporter for the considerable conformational lability of NCp7. © 2008 Wiley Periodicals, Inc. Biopolymers 89: 1125–1135, 2008.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com