Synthetic Transmembrane Peptide Pores for the Single-Molecule Sensing, Application No: 201941014383.
Title
Building sophisticated transmembrane pores for nanotechnology and medicine
Background of the invention
Protein nanopores have been used for the characterization of the size and chemical composition of macromolecules. β-barrels have been engineered extensively, but transmembrane pores based on α-helices are underexplored. Large stable synthetic transmembrane peptide pores formed from short synthetic peptides have not been demonstrated previously. Here, we form a transmembrane pore from 40-amino-acid α-helical peptides based on the porinACj of the Corynebacterium jeikeium. By high resolution single-channel electrical recording, we define the structural properties of the pore and elucidate its assembly pathway. The peptide pore is ion-selective, functional and capable of conducting ions and binding blockers such as cyclic sugars. Our findings have important implications in biotechnology for the design of pores for applications in nanopore technology.
Technology
We emphasize that this is the first large synthetic transmembrane pore built entirely from short synthetic α-helical peptides. 1) We explored here the formation of a stable uniform pore from de novo designed peptides. 2) The structural properties of the pore, including subunit composition. 3) The functional properties of the pore by blocking with cyclodextrins. 4) The assembly pathway of the pore.
No one has assembled such a large stable synthetic transmembrane pore with a single oligomerization and a steady large-conductance state. Often when these experiments are tried, many configurations and oligomerization states are formed, making it difficult to engineer such systems for specificity and distinct functions. The peptide pore developed here is a highly original system due to the unique protein architecture, the significance of transmembrane pores, and their potential applications for DNA sequencing. Additionally, our findings shed light on the mechanism of action of antimicrobial peptides.
The peptide pores are made using chemical synthesis. They are functional and conductive to ions. Importantly, they are ion-selective. Our WT type pore is anion-selective that is blocked by the anionic cyclodextrins. Further, we tuned the selectivity of the pore to conduct specific ions. For example, the mutant pore is cation-selective. Our peptide pore should be of broad interest to membrane protein engineers, especially those looking to build new peptide-based sensors that selectively conduct ions. They also form large pores of 1.4 nm in the lipid membrane with a steady large-conductance state of 4.0 nS, allowing single-molecule sensing of a wide range of analytes.
Significant Merits
We developed self-assembled alpha-helical pores as nanopore sensors. Self-assembly in our context refers to a pore formation process that does not require additional protein domains. This will allow us to create kinetically stable uniform pores in the membranes with a single oligomerization state for the single-molecule sensing. We can vary the subunit stoichiometry and hence the diameter of the alpha-helical pore by using organic templates with different symmetries due to the flexible structure of alpha-helical pores. We can produce large-diameter pores that are appropriate for large analytes, such as antibodies and amyloid structures.
The alpha-helical peptide-based sensors will open up new markets in personalized diagnosis with portable devices for nucleic acid sequencing. In the future, peptide sequencing and sugar sequencing can also be applied.
Related publications
- Krishnan RS, Puthumadathil N, Shaji A, Kumar KS, Mohan G, Mahendran KR*. Designed alpha-helical barrels for charge-selective peptide translocation. Chemical Science. 2021. 12, 639-649.
- Mahendran KR*. Design and Assembly of Transmembrane Helix Barrel. (On invitation, Upcoming scientist). Journal of Membrane Biology. 2020. 253, 491–497.
- Krishnan R S, Satheesan R, Puthumadathil N, Kumar KS, Jayasree P, Mahendran KR*. Autonomously Assembled Synthetic Transmembrane Peptide Pore. Journal of the American Chemical Society. 2019. 141(7):2949-2959.