Abstract：
Nanopore sensing is a label-free single-molecule technique that enables the study of the dynamical structural properties of proteins. Here, we detect the translocation of cytochrome c (Cyt c) through an asymmetric thin nanopore with photothermal heating to evaluate the influence of temperature on Cyt c conformation during its translocation in an electric field. Before Cyt c translocates through an asymmetric thin SiNx nanopore, ∼1 ms trapping events occur due to electric field-induced denaturation. These trapping events were corroborated by a control analysis with a transmission electron microscopy-drilled pore and denaturant buffer. Cyt c translocation events exhibited markedly greater broad current blockade when the pores were photothermally heated. Collectively, our molecular dynamics simulation predicted that an increased temperature facilitates denaturation of the α-helical structure of Cyt c, resulting in greater blockade current during Cyt c trapping. Our photothermal heating method can be used to study the influence of temperature on protein conformation at the single-molecule level in a label-free manner.
摘要：
纳米孔传感是一种无标记的单分子技术，可以研究蛋白质的动态结构特性。这里,我们通过光热加热检测细胞色素c（Cytc）通过不对称薄纳米孔的易位，以评估温度在电场中易位过程中对Cytc构象的影响。在Cytc通过不对称的薄SiNx纳米孔转移之前，由于电场诱导的变性，发生了1毫秒的捕获事件。通过透射电子显微镜钻孔和变性剂缓冲液的对照分析证实了这些捕集事件。当孔被光热加热时，Cytc易位事件表现出明显更大的宽电流阻滞。总的来说，我们的分子动力学模拟预测，温度升高有利于Cytc的α-螺旋结构变性，导致Cytc捕获过程中更大的阻塞电流。我们的光热加热方法可用于以无标记的方式在单分子水平上研究温度对蛋白质构象的影响。
