1.D.51 The Protein Nanopore (ProNP) Family
Despite its successes to probe the chemical reactions and dynamics of macromolecules on sub-millisecond time and nanometer length scales, a major impasse faced by nanopore technology is the need to cheaply and controllably modulate macromolecular capture and trafficking across the nanopore. Asandei et al. 2015 demonstrated that tunable charge separation, engineered at the both ends of a macromolecule, modulates the dynamics of macromolecule capture and traffic through a nanometer-sized pore. They employed a 36 amino acid long peptide containing at its N- and C-termini uniform patches of glutamic acids and arginines, flanking a central segment of asparagines, and studied (1) its capture by the alpha-hemolysin (alpha-HL) and (2) the mean residence time inside the pore, in the presence of a pH gradient across the protein. They proposed a solution to effectively control the dynamics of peptide interactions with the nanopore, with both association and dissociation reaction rates of peptide-α-HL interactions spanning orders of magnitude depending upon solution acidity on the peptide-addition side as well as the transmembrane electric potential (Asandei et al. 2015).
Protein-polymer conjugates, obtained by grafting from the surface of proteins, have been used to generate nanopores. One such protein is the ferric hydroxamate uptake protein component A (FhuA; a beta-barrel transmembrane protein of Escherichia coli). As the lysine residues of naturally occurring FhuA are distributed over the whole protein, FhuA was reengineered to have up to 11 lysines, distributed symmetrically in a rim on the membrane exposed side (outside) of the protein channel, above the hydrophobic region (Charan et al. 2016). Reengineering FhuA ensures a polymer growth only on the outside of the beta-barrel and prevents blockage of the channel as a result of polymerization. A water-soluble initiator for controlled radical polymerization (CRP) was consecutively linked to the lysine residues of FhuA. Such conjugates combine the specific functions of the transmembrane proteins, like maintaining membrane potential gradients or translocation of substrates, with the unique properties of synthetic polymers such as temperature and pH stimuli handles. These conjugates serve as functional nanosized building blocks for applications in targeted drug delivery, self-assembly systems, functional membranes and transmembrane protein gated nanoreactors.
Fragaceatoxin C (FraC), an α-helical pore-forming toxin from an actinoporin protein family, can be reconstituted in sphingomyelin-free standard planar lipid bilayers. Wloka et al. 2016 engineered FraC for DNA analysis and show that the funnel-shaped geometry allows tight wrapping around single-stranded DNA (ssDNA), resolving between homopolymeric C, T, and A polynucleotide stretches. Despite the 1.2 nm internal constriction of FraC, double-stranded DNA (dsDNA) can translocate through the nanopore at high applied potentials, presumably through the deformation of the α-helical transmembrane region of the pore. Therefore, FraC nanopores may be useful for DNA sequencing and dsDNA analysis.
Proteins are the most versatile sources of nanopores, based on our ability to engineer them with sub-nanometer precision (Ayub and Bayley 2016). Novel pores include unnatural amino acid mutagenesis and the application of selection techniques. The diversity of structures has been increased through the development of helix- based pores as well as beta barrels. Therre are also truncated pores, which pierce bilayers through lipid rearrangement, and hybrid pores, which do away with bilayers altogether. Pore dimers, which span two lipid bilayers, have been constructed, and pores based on DNA nanostructures are gaining in importance. While nanopore DNA sequencing has received much attention, protein pores have a wider range of potential applications, requiring specifications that will require engineering efforts to continue for years to come (Ayub and Bayley 2016).