Cressiot Benjamin, Braselmann Esther, Oukhaled Abdelghani, Elcock Adrian H, Pelta Juan, Clark Patricia L
Department of Chemistry & Biochemistry, University of Notre Dame , Notre Dame, Indiana 46556, United States.
LAMBE UMR 8587 CNRS, University of Cergy-Pontoise , Cergy-Pontoise, France.
ACS Nano. 2015 Sep 22;9(9):9050-61. doi: 10.1021/acsnano.5b03053. Epub 2015 Aug 28.
To evaluate the physical parameters governing translocation of an unfolded protein across a lipid bilayer, we studied protein transport through aerolysin, a passive protein channel, at the single-molecule level. The protein model used was the passenger domain of pertactin, an autotransporter virulence protein. Transport of pertactin through the aerolysin nanopore was detected as transient partial current blockades as the unfolded protein partially occluded the aerolysin channel. We compared the dynamics of entry and transport for unfolded pertactin and a covalent end-to-end dimer of the same protein. For both the monomer and the dimer, the event frequency of current blockades increased exponentially with the applied voltage, while the duration of each event decreased exponentially as a function of the electrical potential. The blockade time was twice as long for the dimer as for the monomer. The calculated activation free energy includes a main enthalpic component that we attribute to electrostatic interactions between pertactin and the aerolysin nanopore (despite the low Debye length), plus an entropic component due to confinement of the unfolded chain within the narrow pore. Comparing our experimental results to previous studies and theory suggests that unfolded proteins cross the membrane by passing through the nanopore in a somewhat compact conformation according to the "blob" model of Daoud and de Gennes.
为了评估控制未折叠蛋白跨脂质双层转运的物理参数，我们在单分子水平上研究了蛋白质通过气单胞菌溶素（一种被动蛋白通道）的转运。所使用的蛋白质模型是百日咳杆菌黏附素（一种自转运毒力蛋白）的乘客结构域。当未折叠的蛋白质部分阻塞气单胞菌溶素通道时，百日咳杆菌黏附素通过气单胞菌溶素纳米孔的转运被检测为瞬态部分电流阻断。我们比较了未折叠的百日咳杆菌黏附素及其相同蛋白质的共价端对端二聚体的进入和转移动力学。对于单体和二聚体，电流阻断的事件频率均随施加电压呈指数增加，而每个事件的持续时间则随电势呈指数下降。二聚体的阻断时间是单体的两倍。计算得到的活化自由能包括一个主要的焓成分，我们将其归因于百日咳杆菌黏附素与气单胞菌溶素纳米孔之间的静电相互作用（尽管德拜长度较低），以及由于未折叠链在狭窄孔内的受限而产生的熵成分。将我们的实验结果与先前的研究和理论进行比较表明，根据Daoud和de Gennes的“斑点”模型，未折叠的蛋白质以某种紧密构象穿过纳米孔从而穿过膜。
