Complexity of the folding transition of the B domain of protein A revealed by the high-speed tracking of single-molecule fluorescence time series
Complexity of the folding transition of the B domain of protein A revealed by the high-speed tracking of single-molecule fluorescence time series
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单分子荧光时间序列高速追踪揭示A蛋白B结构域折叠转变的复杂性
DOI：
10.1021/acs.jpcb.5b00414
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发表时间：
2015
期刊：
影响因子：
3.3
通讯作者：
Satoshi Takahashi
中科院分区：
文献类型：
--
作者：
Hiroyuki Oikawa;Kiyoto Kamagata;Munehito Arai;Satoshi Takahashi
利用基于快速流动样品线共聚焦检测的单分子荧光光谱，研究了蛋白质A的B结构域的平衡展开转变。在荧光共振能量转移(FRET)的单分子时间序列测量中，该方法达到了12 0μS的时间分辨率和几毫秒的观测时间。制备了供体和受体双标记的BdpA样品，第一个样品在第22和55位含有荧光团(样品1)，第二个样品在第5和55位(样品2)。通过整体测量监测了氯化胍(GdmCl)诱导的平衡去折叠转变，证明两种样品均服从表观两态去折叠。在没有GdmCl的情况下，两个样品的单分子FRET测量都显示出一个可归属于本征状态(N)的单峰。随着GdmCl浓度的增加，N的FRET效率向较低的值移动，表明本征结构发生了膨胀。当GdmCl浓度较高时，两种样品都转变为未折叠状态(U)。在样品1的展开中点附近，N和U之间的动力学交换导致两个态的平均值和较高的相对涨落。U中不同分子的时间序列显示出略有不同的FRET效率，表明存在明显的非均质性。因此，对单分子荧光信号的高速跟踪揭示了隐藏在蛋白质折叠的明显两态行为中的复杂性和异质性。
The equilibrium unfolding transition of the B domain of protein A (BdpA) was investigated by using single-molecule fluorescence spectroscopy based on line-confocal detection of fast-flowing samples. The method achieved the time resolution of 120 μs and the observation time of a few milliseconds in the single-molecule time-series measurements of fluorescence resonance energy transfer (FRET). Two samples of BdpA doubly labeled with donor and acceptor fluorophores, the first possessing fluorophores at residues 22 and 55 (sample 1) and the second at residues 5 and 55 (sample 2), were prepared. The equilibrium unfolding transition induced by guanidium chloride (GdmCl) was monitored by bulk measurements and demonstrated that the both samples obey the apparent two-state unfolding. In the absence of GdmCl, the single-molecule FRET measurements for the both samples showed a single peak assignable to the native state (N). The FRET efficiency for N shifts to lower values as the increase of GdmCl concentration, suggesting the swelling of the native state structure. At the higher concentration of GdmCl, the both samples convert to the unfolded state (U). Near the unfolding midpoint for sample 1, the kinetic exchange between N and U causes the averaging of the two states and the higher values of the relative fluctuation. The time series for different molecules in U showed slightly different FRET efficiencies, suggesting the apparent heterogeneity. Thus, the high-speed tracking of fluorescence signals from single molecules revealed a complexity and heterogeneity hidden in the apparent two-state behavior of protein folding.