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 the 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. There 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). Natural receptors in biomembranes can be used for designing biosensing methods (Sugawara 2017). Protein nanopores have been used for a variety of sensing applications (Zhang et al. 2022).
Monitoring current flow through a single nanopore has proven to be a powerful technique. Transmembrane proteins, such as α-hemolysin, provide attractive platforms for nanopore sensing applications due to their precise structures. However, many nanopore applications require the introduction of functional groups to tune selectivity. Borsley and Cockroft 2017 demonstrated the in situ synthetic modification of a wild-type α-hemolysin nanopore embedded in a membrane. They showed that reversible dynamic covalent iminoboronate formation and the resulting changes in the ion current flowing through an individual nanopore can be used to map the reactive behavior of lysine residues within the nanopore channel. Modification of lysine residues located outside the nanopore channel was found not to affect the stability or utility of the nanopore. Protein ligand-induced amplification in the 1/f noise of a protein-selective nanopore has been reported (Sun et al. 2020). Structural determinants of chirally selective transport of amino acids through the alpha-hemolysin protein nanopores of free-standing planar lipid membranes has been characterized (Lee et al. 2024).
There is a pressing need for the creation of nanopores equipped with relatively large functional groups for the sampling of biomolecular events on their extramembranous side. Larimi et al. 2021 designed, produced, and analyzed protein nanopores encompassing a robust truncation of a monomeric beta-barrel membrane protein. An exogenous stably folded protein was anchored within the aqueous phase via a flexible peptide tether of varying length. They examined the pore-forming properties of these modular protein nanopores using protein engineering and single- molecule electrophysiology. This revealed distinctions in the nanopore conductance and current fluctuations that arose from tethering the exogenous protein to either the N-terminus or the C-terminus. These nanopores insert into a planar lipid membrane with one specific conductance among a set of three substate conductance values. The occurrence probabilities of these insertion substates depend on the length of the peptide tether, the orientation of the exogenous protein with respect to the nanopore opening, and the molecular mass of tethered protein (Larimi et al. 2021).
Filtration through membranes with nanopores is typically associated with high transmembrane pressures and high energy consumption. This problem can be addressed by reducing the respective membrane thickness. Schwieters et al. 2021 described a simple procedure for the preparation of ultrathin membranes based on protein nanopores, which exhibit excellent water permeance, two orders of magnitude superior to comparable, industrially applied membranes. Incorporation of either closed or open protein nanopores allowed tailoring the membrane's ion permeability. To form such membranes, the transmembrane protein, ferric hydroxamate uptake protein component A (FhuA), or its open-pore variant were assembled at the air-water interface of a Langmuir trough, compressed to a dense film, crosslinked by glutaraldehyde, and transferred to various support materials (Schwieters et al. 2021).
A general approach to the design of transmembrane beta-barrel (TMB) pores with different diameters and pore geometries has been presented (Berhanu et al. 2023). NMR and crystallographic characterization showed that the designs are stably folded with structures close to the design models. The authors reported the first examples of de novo designed TMBs with 10, 12 and 14 stranded beta-barrels. The designs have distinct conductances that correlate with their pore diameter, ranging from 110 pS (~0.5 nm pore diameter) to 430 pS (~1.1 nm pore diameter), and can be converted into sensitive small-molecule sensors with high signal to noise ratios. The capability to generate on demand β-barrel pores of defined geometry opens up fundamentally new opportunities for custom engineering of sequencing and sensing technologies (Berhanu et al. 2023).