Department of Electrical Engineering and ‡Departments of Electrical Engineering and Biological Science, University of Notre Dame , Notre Dame, Indiana 46556, United States.
ACS Nano. 2017 Jun 27;11(6):5440-5452. doi: 10.1021/acsnano.6b08452. Epub 2017 Jun 2.
It is now possible to create, in a thin inorganic membrane, a single, sub-nanometer-diameter pore (i.e., a sub-nanopore) about the size of an amino acid residue. To explore the prospects for sequencing protein with it, measurements of the force and current were performed as two denatured histones, which differed by four amino acid residue substitutions, were impelled systematically through the sub-nanopore one at a time using an atomic force microscope. The force measurements revealed that once the denatured protein, stabilized by sodium dodecyl sulfate (SDS), translocated through the sub-nanopore, a disproportionately large force was required to pull it back. This was interpreted to mean that the SDS was cleaved from the protein during the translocation. The force measurements also exposed a dichotomy in the translocation kinetics: either the molecule slid nearly frictionlessly through the pore or it slipped-and-stuck. When it slid frictionlessly, regardless of whether the molecule was pulled N-terminus or C-terminus first through the pore, regular patterns were observed intermittently in the force and blockade current fluctuations that corresponded to the distance between stretched residues. Furthermore, the amplitude of the fluctuations in the current blockade were correlated with the occluded volume associated with the amino acid residues in the pore. Finally, a comparison of the patterns in the current fluctuations associated with the two practically identical histones supported the conclusion that a sub-nanopore was sensitive enough to discriminate amino acid substitutions in the sequence of a single protein molecule by measuring volumes of 0.1 nm per read.
现在可以在薄的无机膜中创建一个单一的、亚纳米直径的孔（即亚纳米孔），其大小与氨基酸残基相当。为了探索用它对蛋白质进行测序的前景，我们对力和电流进行了测量，将两种变性组蛋白（它们在四个氨基酸残基上有所不同）一次一个地通过原子力显微镜系统地推动穿过亚纳米孔。力的测量结果表明，一旦变性蛋白被十二烷基硫酸钠（SDS）稳定，通过亚纳米孔迁移时，需要施加不成比例的力才能将其拉回。这意味着 SDS 在转运过程中从蛋白质上被切断。力的测量还揭示了转运动力学的二分法：要么分子几乎无摩擦地滑过孔，要么滑动-卡住。当它无摩擦地滑动时，无论分子是先被 N 端还是 C 端拉过孔，在力和阻断电流波动中都会间歇性地观察到与伸展残基之间距离相对应的规则模式。此外，电流阻断波动的幅度与孔内氨基酸残基的封闭体积相关。最后，对两种几乎相同的组蛋白相关的电流波动模式进行比较，支持了这样的结论：亚纳米孔足够灵敏，可以通过测量每个读取 0.1nm 的体积来区分单个蛋白质分子序列中的氨基酸取代。
