Sensing Native Protein Solution Structures Using a Solid-state Nanopore: Unraveling the States of VEGF


Abstract Monitoring individual proteins in solution while simultaneously obtaining tertiary and quaternary structural information is challenging. In this study, translocation of the vascular endothelial growth factor (VEGF) protein through a solid-state nanopore (ssNP) produces distinct ion-current blockade amplitude levels and durations likely corresponding to monomer, dimer, and higher oligomeric states. Upon changing from a non-reducing to a reducing condition, ion-current blockage events from the monomeric state dominate, consistent with the expected reduction of the two inter-chain VEGF disulfide bonds. Cleavage by plasmin and application of either a positive or a negative NP bias results in nanopore signals corresponding either to the VEGF receptor recognition domain or to the heparin binding domain, accordingly. Interestingly, multi-level analysis of VEGF events reveals how individual domains affect their translocation pattern. Our study shows that careful characterization of ssNP results elucidates real-time structural information about the protein, thereby complementing classical techniques for structural analysis of proteins in solution with the added advantage of quantitative single-molecule resolution of native proteins

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oai:doaj.org/article:c01e7ff0ef6246de8a75dd3501511dceLast time updated on 6/4/2019

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