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

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 <i>c</i> (Cyt <i>c</i>) through an asymmetric thin nanopore with photothermal heating to evaluate the influence of temperature on Cyt <i>c</i> conformation during its translocation in an electric field. Before Cyt <i>c</i> translocates through an asymmetric thin SiN_<i>x</i> 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 <i>c</i> 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 <i>c</i>, resulting in greater blockade current during Cyt <i>c</i> 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.