Oikawa Hiroyuki, Kamagata Kiyoto, Arai Munehito, Takahashi Satoshi
†Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1 Katahira, Aoba, Sendai, Miyagi 980-8577, Japan.
‡Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan.
J Phys Chem B. 2015 May 21;119(20):6081-91. doi: 10.1021/acs.jpcb.5b00414. Epub 2015 May 8.
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.
利用基于快速流动样品线共聚焦检测的单分子荧光光谱法，研究了蛋白A的B结构域（BdpA）的平衡去折叠转变。在荧光共振能量转移（FRET）的单分子时间序列测量中，该方法实现了120微秒的时间分辨率和几毫秒的观测时间。制备了用供体和受体荧光团双标记的两个BdpA样品，第一个样品在第22和55位残基处带有荧光团（样品1），第二个样品在第5和55位残基处带有荧光团（样品2）。通过体相测量监测了由氯化胍（GdmCl）诱导的平衡去折叠转变，结果表明两个样品均遵循明显的两态去折叠。在没有GdmCl的情况下，两个样品的单分子FRET测量均显示出一个可归因于天然态（N）的单峰。随着GdmCl浓度的增加，N的FRET效率向较低值移动，表明天然态结构发生膨胀。在较高的GdmCl浓度下，两个样品均转变为未折叠态（U）。在样品1的去折叠中点附近，N和U之间的动力学交换导致两种状态的平均化以及相对波动的较高值。U中不同分子的时间序列显示出略有不同的FRET效率，表明存在明显的异质性。因此，对单分子荧光信号的高速跟踪揭示了蛋白质折叠明显的两态行为中隐藏的复杂性和异质性。
