Direct Measurement of Single-Molecule Adenosine Triphosphatase Hydrolysis Dynamics

作者：Jie Li, Gen He, Hiroshi Ueno, Wenzhe Liu, Hiroyuki Noji, Chuanmin Qi, Xuefeng Guo · 发表于：ACS Nano · 年份：2017 · DOI：10.1021/acsnano.7b07639 · 被引用次数：23 · 研究领域：ATP Synthase and ATPases Research、Advanced Electron Microscopy Techniques and Applications、Nanopore and Nanochannel Transport Studies

F 1 -ATPase (F 1 ) is a bidirectional molecular motor that hydrolyzes nearly all ATPs to fuel the cellular processes. Optical observation of labeled F 1 rotation against the α 3 β 3 hexamer ring revealed the sequential mechanical rotation steps corresponding to ATP binding/ADP release and ATP hydrolysis/Pi release. These substeps originate from the F 1 rotation but with heavy load on the γ shaft due to fluorescent labeling and the photophysical limitation of an optical microscope, which hampers better understanding of the intrinsic kinetic behavior of ATP hydrolysis. In this work, we present a method capable of electrically monitoring ATP hydrolysis of a single label-free F 1 in real time by using a high-gain silicon nanowire-based field-effect transistor circuit. We reproducibly observe the regular current signal fluctuations with two distinct levels, which are induced by the binding dwell and the catalytic dwell, respectively, in both concentration- and temperature-dependent experiments. In comparison with labeled F 1, the hydrolysis rate of nonlabeled F 1 used in this study is 1 order of magnitude faster (1.69 × 10 8 M –1 s –1 at 20 °C), and the differences between two sequential catalytic rates are clearer, demonstrating the ability of nanowire nanocircuits to directly probe the intrinsic dynamic processes of the biological activities with single-molecule/single-event sensitivity. This approach is complementary to traditional optical methods, offering endless opportunitie...