Single-Molecule Titration in a Protein Nanoreactor Reveals the Protonation/Deprotonation Mechanism of a C:C Mismatch in DNA.
Single-Molecule Titration in a Protein Nanoreactor Reveals the Protonation/Deprotonation Mechanism of a C:C Mismatch in DNA.
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DOI：
10.1021/jacs.8b00593
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发表时间：
2018-04-18
影响因子：
15
通讯作者：
White HS
中科院分区：
文献类型：
--
作者：
Ren H;Cheyne CG;Fleming AM;Burrows CJ;White HS
单分子反应的测量可以阐明常常隐藏在系综分析之外的微观机制。在这里，我们报道了限制在α-溶血素(α-HL)纳米孔中长达3小时的单个DNA二聚体的酸碱滴定，同时监测通过纳米孔的离子电流。对于在α-HL的锁存收缩附近包含C：C失配的双工，电流-时间迹线中的两个状态之间的调制归因于C：C失配的基翻转。随着pH的降低，C：C失配从螺旋内态翻转到螺旋外状态的速率(kintra-Extra)降低，而碱基从螺旋外状态翻转到螺旋内状态的速率(kExtra-Intra)保持不变。Kintra-Extra和kExtra-Intra都在10−2 S−1到10−1 S−1的数量级上，并在测量的时间范围内保持稳定(几个小时)。用隐马尔可夫动力学模型分析碱基翻转的pH依赖动力学表明，当碱基对处于螺旋内状态时，质子化/去质子化发生。我们还证明了质子化的速度受到H+向α-HL纳米孔的传输的限制。单分子动力学同位素实验表明，运动外(KH/Kd~5)有很大的动力学同位素效应(Kie)，而Kh/Kd~1.2内有有限的Kie，支持我们的模型。我们的实验相当于使用纳米孔进行的最长的单分子测量，并展示了它在询问受限几何中的单分子反应机理方面的应用。
Measurement of single-molecule reactions can elucidate microscopic mechanisms that are often hidden from ensemble analysis. Herein, we report the acid-base titration of a single DNA duplex confined within the α-hemolysin (α-HL) nanopore for up to 3 h, while monitoring the ionic current through the nanopore. Modulation between two states in the current-time trace for duplexes containing the C:C mismatch in proximity to the latch constriction of α-HL is attributed to the base flipping of the C:C mismatch. As the pH is lowered, the rate for the C:C mismatch to flip from the intra-helical state to the extra-helical state (kintra-extra) decreases, while the rate for base flipping from the extra-helical state to the intra-helical state (kextra-intra) remains unchanged. Both kintra-extra and kextra-intra are on the order of 10−2 s−1 to 10−1 s−1 and remain stable over the timescale of the measurement (several hours). Analysis of the pH-dependent kinetics of base flipping using a hidden Markov kinetic model demonstrates that protonation/deprotonation occurs while the base pair is in the intra-helical state. We also demonstrate that the rate of protonation is limited by transport of H+ into the α-HL nanopore. Single-molecule kinetic isotope experiments exhibit a large kinetic isotope effect (KIE) for kintra-extra (kH/kD ~ 5) but a limited KIE for kextra-intra (kH/kD ~ 1.2), supporting our model. Our experiments correspond to the longest single-molecule measurements performed using a nanopore, and demonstrate its application in interrogating mechanisms of single-molecule reactions in confined geometries.
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DOI：
10.1038/nsb902
发表时间：
2003-03-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
--
作者：
Fromme, JC;Bruner, SD;Verdine, GL
通讯作者：
Verdine, GL
影响因子：
3.4
作者：
Noskov, SY;Im, W;Roux, B
通讯作者：
Roux, B
影响因子：
3.4
作者：
Aksimentiev, A;Schulten, K
通讯作者：
Schulten, K
DOI：
10.1073/pnas.0502947102
发表时间：
2005-08-30
影响因子：
11.1
作者：
Mathé, J;Aksimentiev, A;Meller, A
通讯作者：
Meller, A
影响因子：
16.6
作者：
EIGEN, M
通讯作者：
EIGEN, M