Significant Heterogeneity and Slow Dynamics of the Unfolded Ubiquitin Detected by the Line Confocal Method of Single-Molecule Fluorescence Spectroscopy
Significant Heterogeneity and Slow Dynamics of the Unfolded Ubiquitin Detected by the Line Confocal Method of Single-Molecule Fluorescence Spectroscopy
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单分子荧光光谱线共焦法检测未折叠泛素的显着异质性和缓慢动态
DOI：
10.1021/acs.jpcb.6b05481
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发表时间：
2016
期刊：
影响因子：
3.3
通讯作者：
Satoshi Takahashi
中科院分区：
文献类型：
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作者：
Masataka Saito;Supawich Kamonprasertsuk;Satomi Suzuki;Kei Nanatani;Hiroyuki Oikawa;Keiichiro Kushiro;Madoka Takai;Po-ting Chen;Eric H.-L. Chen;Rita P.-Y. Chen; Satoshi Takahashi
利用单分子荧光光谱研究了Alexa 488和Alexa 647区域特异性双标记泛素的非折叠态构象和动力学。与传统的单分子方法相比，线性共聚焦荧光检测系统结合快速的样品流动，能够以改进的结构和时间分辨率表征未折叠蛋白质。然而，在目前的调查的初始阶段，标记的泛素的单分子Fo Prster共振能量转移（sm-FRET）数据是有缺陷的工件所造成的吸附样品的表面的熔融石英流动芯片和样品输送系统。2-甲基丙烯酰氧基乙基磷酰胆碱聚合物的共价涂层的流动芯片表面被发现抑制的文物。基于涂层流动芯片的sm-FRET测量表明，天然条件下泛蛋白的sm-FRET效率的直方图分布较窄，与散粒噪声预期的概率密度函数（PDF）相当，证明了结构的同质性天然状态。相比之下，在100 μs的时间分辨率下获得的未折叠泛素的sm-FRET效率的直方图的分布明显比从散粒噪声预期的PDF更宽，证明了未折叠状态构象的异质性。的sm-FRET效率的展开状态的各种保持，即使在评估的移动平均值的痕迹与窗口大小为1毫秒，这表明异构构象的构象平均大多发生在时间域慢于1毫秒。标记的荧光团周围的局部结构的异质性被推断为结构异质性的原因。非折叠蛋白质的异质性和缓慢的动力学特性可能是未折叠蛋白质的共同特性。
The conformation and dynamics of the unfolded state of ubiquitin doubly labeled regiospecifically with Alexa488 and Alexa647 were investigated using single-molecule fluorescence spectroscopy. The line confocal fluorescence detection system combined with the rapid sample flow enabled the characterization of unfolded proteins at the improved structural and temporal resolutions compared to the conventional single-molecule methods. In the initial stage of the current investigation, however, the single-molecule Förster resonance energy transfer (sm-FRET) data of the labeled ubiquitin were flawed by artifacts caused by the adsorption of samples to the surfaces of the fused-silica flow chip and the sample delivery system. The covalent coating of 2-methacryloyloxyethyl phosphorylcholine polymer to the flow chip surface was found to suppress the artifacts. The sm-FRET measurements based on the coated flow chip demonstrated that the histogram of the sm-FRET efficiencies of ubiquitin at the native condition were narrowly distributed, which is comparable to the probability density function (PDF) expected from the shot noise, demonstrating the structural homogeneity of the native state. In contrast, the histogram of the sm-FRET efficiencies of the unfolded ubiquitin obtained at a time resolution of 100 μs was distributed significantly more broadly than the PDF expected from the shot noise, demonstrating the heterogeneity of the unfolded state conformation. The variety of the sm-FRET efficiencies of the unfolded state remained even after evaluating the moving average of traces with a window size of 1 ms, suggesting that conformational averaging of the heterogeneous conformations mostly occurs in the time domain slower than 1 ms. Local structural heterogeneity around the labeled fluorophores was inferred as the cause of the structural heterogeneity. The heterogeneity and slow dynamics revealed by the line confocal tracking of sm-FRET might be common properties of the unfolded proteins.