Improving insertions of mutated G20C portal protein into lipid bilayers for nanopore sensing.

Abstract

In recent years, the third-generation nanopore sequencing technology (nanopore sequencing) has stood out due to its long read lengths, simple operation, portability, and real-time sequence readout, and has also shown good prospects in biomolecular detection. Mainstream nanopore architectures implement transmembrane proteins that insert into an isolative membrane, where the signal resolution of the nanopore is dominated by the nature of the pore protein. Current choices focus on naturally bio-membrane dwelling proteins, whereas a broad range of other proteins may also provide pores suitable for nanopore sensing, although their implementation has been limited by a poor ability to insert into organic membranes. Here, we describe a strategy for inserting viral portal proteins with flexible inner pores into lipid membranes: We expressed different mutants of G20C portal protein to 1) enable chemical modifications of hydrophobic anchors to the portal's exterior structure and 2) substitute hydrophobic amino acid residues into the portal's base in order to facilitate membrane insertion. In this work, we engineered and purified different portal mutants, and verified their structural integrity with electron microscopy. Building on our recent demonstration of DNA sensing with G20C mutants, we are in the process of characterizing the sensing capabilities of these mutants through ionic current recordings of DNA and peptide translocation through the portal mutant. We expect to apply this approach to a wide range of non-membrane, ring-shaped proteins and facilitate the development of new biological nanopores that can provide improved signal resolution and data quality.