Force-dependent chemical kinetics of disulfide bond reduction observed with single-molecule techniques.
Force-dependent chemical kinetics of disulfide bond reduction observed with single-molecule techniques.
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DOI：
10.1073/pnas.0511035103
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发表时间：
2006-05
影响因子：
11.1
通讯作者：
A. Wiita;Sri Rama Koti Ainavarapu;Hector H. Huang;Julio M. Fernandez
中科院分区：
文献类型：
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作者：
A. Wiita;Sri Rama Koti Ainavarapu;Hector H. Huang;Julio M. Fernandez
机械力调节化学反应动力学的机制尚不清楚。在这里，我们使用单分子力钳光谱和蛋白质工程研究力对巯基/二硫键交换动力学的影响。通过硫醇/二硫键交换化学反应还原二硫键在调节蛋白质功能中至关重要，并且已知在机械应激蛋白质中发生。我们对单个工程二硫键施加恒定的拉伸力，并通过DTT测量其还原速率。虽然减少率是线性依赖于DTT的浓度，它是指数依赖于所施加的力，增加10倍以上的300 pN的范围。该结果预测，在硫醇/二硫键交换反应的过渡态，二硫键延长0.34 A。我们在单键水平上的工作直接证明了蛋白质中的巯基/二硫键交换是一种力依赖的化学反应。我们的研究结果表明，机械力在体内二硫键还原中起作用，这是传统生物化学从未探索过的特性。此外，我们的工作还表明，任何化学反应，导致键延长的动力学将是力依赖性的。
The mechanism by which mechanical force regulates the kinetics of a chemical reaction is unknown. Here, we use single-molecule force-clamp spectroscopy and protein engineering to study the effect of force on the kinetics of thiol/disulfide exchange. Reduction of disulfide bonds through the thiol/disulfide exchange chemical reaction is crucial in regulating protein function and is known to occur in mechanically stressed proteins. We apply a constant stretching force to single engineered disulfide bonds and measure their rate of reduction by DTT. Although the reduction rate is linearly dependent on the concentration of DTT, it is exponentially dependent on the applied force, increasing 10-fold over a 300-pN range. This result predicts that the disulfide bond lengthens by 0.34 A at the transition state of the thiol/disulfide exchange reaction. Our work at the single bond level directly demonstrates that thiol/disulfide exchange in proteins is a force-dependent chemical reaction. Our findings suggest that mechanical force plays a role in disulfide reduction in vivo, a property that has never been explored by traditional biochemistry. Furthermore, our work also indicates that the kinetics of any chemical reaction that results in bond lengthening will be force-dependent.