BPS2026 – Modification of β-barrel nanopores and reconstitution via membrane fusion between nanodiscs and lipid vesicles

Protein nanopores in cell membranes allow the penetration of substances such as ions, nutrients, and small molecules. Recently, protein nanopores have been applied as biological sensors and molecular transporters in bilayer lipid membrane (BLM) or liposomes. We successfully modified the pore size of outer membrane protein G (OmpG) by changing the number of β-strands. DNA with different shapes was detected in a BLM system depending on the pore diameter of these mutant OmpGs (Tosaka, T. and Kamiya, K., ACS Appl. Nano Mater ., 2022). Although ions were efficiently transported into GUVs reconstituting the mutant OmpG, fluorescent molecules were scarcely transported (Tosaka, T. and Kamiya, K., ACS Omega , 2024). In this study, to construct a larger pore size using Omp for transporting various molecules, we redesigned the BtuB nanopore with 22 β-strands, derived from wild-type BtuB, known as vitamin B12 transporter, using molecular dynamics simulations and protein structure prediction tools such as ColabFold (a deep learning model) and I-TASSER (an ab initio protein folding prediction tool). The BtuB nanopore was designed by deleting regions of the protein sequence corresponding to the extracellular loops and the inner β-barrel. The BtuB nanopore was expressed and purified from E. coli . The transportation of fluorescent molecules through the BtuB nanopore was investigated using BLM or GUVs fused with nanodiscs containing the BtuB. Membrane fusion was triggered by interactions between the His-tag of the membrane scaffold protein and DGS-NTA(Ni) lipids of BLM and GUVs. Nanopore formation of BtuB was confirmed by electrophysiological measurements using the BLM system. In addition, the transport of fluorescent molecules was observed from the outer aqueous phase to the inner aqueous phase of the GUV via the BtuB nanopore.