Single-Molecule Study on Interactions between Cyclic Nonribosomal Peptides and Protein Nanopore

Nonribosomal peptides (NRPs) are a type of secondary metabolites that mostly originated from microorganisms such as bacteria and fungi. Their proteolytic stability, highly selective bioactivity, and microorganism-specificity have made them an attractive source of drugs for the pharmaceutical industry. Herein, with microcystins (MCs) as a NRP model, we, for the first time, proposed a sensitive method to study the interactions between NRPs and the protein nanopore. Because of the large molecular size (∼3 nm diameter) of MCs and their net negative charges, MCs failed to translocate through the α-hemolysin (α-HL) protein channel. Our results demonstrated that the biomolecular interaction of MC-α-HL protein was significantly affected by the applied potential bias. The constant blockage amplitude in the voltage-dependent studies indicated that the current modulation events were dominantly contributed to the bumping interaction between MCs and the α-HL protein under the electrophoretic force. The mean residence time of the bumping events exhibited a two-stage decrease (from 1.90 to 1.02 ms, and from 1.02 to 0.69 ms) at the threshold voltages of −70 mV and −100 mV, respectively. Using our strategy (i.e., based on their electrophoretic driven interaction with the α-HL protein pore), discrimination of different MC molecules (MC-LR, MC-RR, MC-YR, and linear analog) with varied branched residues could be accomplished. This work should provide insight into developing a rapid and effective method for the identification of cyclic NRPs as valuable biomarkers for fungal infections.