Butler Tom Z, Gundlach Jens H, Troll Mark
Department of Physics, University of Washington, Seattle, Washington, USA.
Biophys J. 2007 Nov 1;93(9):3229-40. doi: 10.1529/biophysj.107.107003. Epub 2007 Aug 3.
We characterize the substate structure of current blockades produced when single-stranded polynucleotide molecules were electrophoretically driven into the alpha-hemolysin protein pore. We frequently observe substates where the ionic current is reduced by approximately 50%. Most of these substates can be associated with a molecular configuration where a polymer occupies only the vestibule region of the pore, though a few appear related to a polymer occupying only the transmembrane beta-barrel region of the pore. The duration of the vestibule configuration depends on polymer composition and on which end of the polymer, 3' or 5', subsequently threads into the narrowest constriction and initiates translocation. Below approximately 140 mV a polymer is more likely to escape from the vestibule against the applied voltage gradient, while at higher voltages a polymer is more likely to follow the voltage gradient by threading through the narrowest constriction and translocating through the pore. Increasing the applied voltage also increases the duration of the vestibule configuration. A semiquantitative model of these trends suggests that escape has stronger voltage dependence than threading, and that threading is sensitive to polymer orientation while escape is not. These results emphasize the utility of alpha-hemolysin as a model system to study biologically relevant physical and chemical processes at the single-molecule level.
我们对单链多核苷酸分子电泳驱动进入α-溶血素蛋白孔时产生的电流阻断亚状态结构进行了表征。我们经常观察到离子电流降低约50%的亚状态。这些亚状态中的大多数可能与聚合物仅占据孔前庭区域的分子构型相关，不过也有一些似乎与聚合物仅占据孔跨膜β桶区域有关。前庭构型的持续时间取决于聚合物组成以及聚合物的哪一端（3'端或5'端）随后穿入最窄的收缩处并启动转运。在约140 mV以下，聚合物更有可能逆着施加的电压梯度从前庭逸出，而在较高电压下，聚合物更有可能通过穿入最窄的收缩处并穿过孔来顺着电压梯度移动。增加施加的电压也会增加前庭构型的持续时间。这些趋势的半定量模型表明，逸出比穿入具有更强的电压依赖性，并且穿入对聚合物取向敏感而逸出不敏感。这些结果强调了α-溶血素作为在单分子水平研究生物学相关物理和化学过程的模型系统的实用性。
