Photothermally Heated Asymmetric Thin Nanopores Suggest\nthe Influence of Temperature on the Intermediate Conformational State\nof Cytochrome <i>c</i> in an Electric Field

Nanopore\nsensing is a label-free single-molecule technique that\nenables the study of the dynamical structural properties of proteins.\nHere, we detect the translocation of cytochrome <i>c</i> (Cyt <i>c</i>) through an asymmetric thin nanopore with\nphotothermal heating to evaluate the influence of temperature on Cyt <i>c</i> conformation during its translocation in an electric field.\nBefore Cyt <i>c</i> translocates through an asymmetric thin\nSiN<sub><i>x</i></sub> nanopore, ∼1 ms trapping events\noccur due to electric field-induced denaturation. These trapping events\nwere corroborated by a control analysis with a transmission electron\nmicroscopy-drilled pore and denaturant buffer. Cyt <i>c</i> translocation events exhibited markedly greater broad current blockade\nwhen the pores were photothermally heated. Collectively, our molecular\ndynamics simulation predicted that an increased temperature facilitates\n denaturation of the α-helical structure of Cyt <i>c</i>, resulting in greater blockade current during Cyt <i>c</i> trapping. Our photothermal heating method can be used to study the\ninfluence of temperature on protein conformation at the single-molecule\nlevel in a label-free manner.