Complex and Non-sequential Current Signatures of a β-Hairpin Peptide Confined in a Nanopore

Overview

Journal Anal Chem

Date 2025 Jan 22

PMID 39841857

Authors

Misa Yamaji

Mauro Chinappi

Blasco Morozzo Della Rocca

Kenji Usui

Ryuji Kawano

Affiliations

Soon will be listed here.

Abstract

Nanopore sensing is widely used for single-molecule detection, originally applied to nucleic acids and now extended to protein sensing. Our study focuses on the complex conformational changes of peptides in nanopores, which may have implications for peptide fingerprinting and protein identification. Specifically, we investigated the interaction of a β-hairpin peptide (SV28) within an α-hemolysin (αHL) nanopore. Our experiments revealed that SV28 is captured via dielectrophoresis and exhibits long dwell times within the nanopore, leading to multiple current blockade levels. Unlike DNA hairpins, the peptide showed non-sequential transitions among four distinct blockade levels. This complex behavior indicates that the peptide dynamics in nanopores cannot be simply modeled along a single reaction coordinate. Our findings provide insights into peptide-nanopore interactions, which are potentially useful for developing nanopore-based peptide identification technologies.

References

Lucas F, Abraham Versloot R, Yakovlieva L, Walvoort M, Maglia G . Protein identification by nanopore peptide profiling. Nat Commun. 2021; 12(1):5795. PMC: 8490355. DOI: 10.1038/s41467-021-26046-9. View

Matsushita M, Shoji K, Takai N, Kawano R . Biological Nanopore Probe: Probing of Viscous Solutions in a Confined Nanospace. J Phys Chem B. 2020; 124(12):2410-2416. DOI: 10.1021/acs.jpcb.9b11096. View

Vercoutere W, Olsen H, Deamer D, Haussler D, Akeson M . Rapid discrimination among individual DNA hairpin molecules at single-nucleotide resolution using an ion channel. Nat Biotechnol. 2001; 19(3):248-52. DOI: 10.1038/85696. View

Warburton P, Sebra R . Long-Read DNA Sequencing: Recent Advances and Remaining Challenges. Annu Rev Genomics Hum Genet. 2023; 24:109-132. DOI: 10.1146/annurev-genom-101722-103045. View

Huang G, Willems K, Soskine M, Wloka C, Maglia G . Electro-osmotic capture and ionic discrimination of peptide and protein biomarkers with FraC nanopores. Nat Commun. 2017; 8(1):935. PMC: 5715100. DOI: 10.1038/s41467-017-01006-4. View

Yu L, Kang X, Li F, Mehrafrooz B, Makhamreh A, Fallahi A . Unidirectional single-file transport of full-length proteins through a nanopore. Nat Biotechnol. 2023; 41(8):1130-1139. PMC: 10329728. DOI: 10.1038/s41587-022-01598-3. View

Zhang M, Tang C, Wang Z, Chen S, Zhang D, Li K . Real-time detection of 20 amino acids and discrimination of pathologically relevant peptides with functionalized nanopore. Nat Methods. 2024; 21(4):609-618. PMC: 11009107. DOI: 10.1038/s41592-024-02208-7. View

Liu A, Zhao Q, Krishantha D, Guan X . Unzipping of Double-stranded DNA in Engineered α-Hemolysin Pores. J Phys Chem Lett. 2011; 2(12):1372-1376. PMC: 3119559. DOI: 10.1021/jz200525v. View

Asandei A, Schiopu I, Chinappi M, Seo C, Park Y, Luchian T . Electroosmotic Trap Against the Electrophoretic Force Near a Protein Nanopore Reveals Peptide Dynamics During Capture and Translocation. ACS Appl Mater Interfaces. 2016; 8(20):13166-79. DOI: 10.1021/acsami.6b03697. View

10.

Ouldali H, Sarthak K, Ensslen T, Piguet F, Manivet P, Pelta J . Electrical recognition of the twenty proteinogenic amino acids using an aerolysin nanopore. Nat Biotechnol. 2019; 38(2):176-181. PMC: 7008938. DOI: 10.1038/s41587-019-0345-2. View

11.

Liu J, Aksimentiev A . Molecular Determinants of Current Blockade Produced by Peptide Transport Through a Nanopore. ACS Nanosci Au. 2024; 4(1):21-29. PMC: 10885333. DOI: 10.1021/acsnanoscienceau.3c00046. View

12.

Varongchayakul N, Song J, Meller A, Grinstaff M . Single-molecule protein sensing in a nanopore: a tutorial. Chem Soc Rev. 2018; 47(23):8512-8524. PMC: 6309966. DOI: 10.1039/c8cs00106e. View

13.

Sun L, Li H, Xu X, Luo M . Simulation study on the translocation of polyelectrolyte through conical nanopores. J Phys Condens Matter. 2018; 30(49):495101. DOI: 10.1088/1361-648X/aaeb19. View

14.

James P, Quadroni M, Carafoli E, Gonnet G . Protein identification by mass profile fingerprinting. Biochem Biophys Res Commun. 1993; 195(1):58-64. DOI: 10.1006/bbrc.1993.2009. View

15.

Gordon L, Haydon D . Kinetics and stability of alamethicin conducting channels in lipid bilayers. Biochim Biophys Acta. 1976; 436(3):541-56. DOI: 10.1016/0005-2736(76)90439-9. View

16.

Akeson M, Branton D, Kasianowicz J, Brandin E, Deamer D . Microsecond time-scale discrimination among polycytidylic acid, polyadenylic acid, and polyuridylic acid as homopolymers or as segments within single RNA molecules. Biophys J. 1999; 77(6):3227-33. PMC: 1300593. DOI: 10.1016/S0006-3495(99)77153-5. View

17.

Jain M, Olsen H, Paten B, Akeson M . The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community. Genome Biol. 2016; 17(1):239. PMC: 5124260. DOI: 10.1186/s13059-016-1103-0. View

18.

Waduge P, Hu R, Bandarkar P, Yamazaki H, Cressiot B, Zhao Q . Nanopore-Based Measurements of Protein Size, Fluctuations, and Conformational Changes. ACS Nano. 2017; 11(6):5706-5716. DOI: 10.1021/acsnano.7b01212. View

19.

Giansanti P, Tsiatsiani L, Low T, Heck A . Six alternative proteases for mass spectrometry-based proteomics beyond trypsin. Nat Protoc. 2016; 11(5):993-1006. DOI: 10.1038/nprot.2016.057. View

20.

Kasianowicz J, Brandin E, Branton D, Deamer D . Characterization of individual polynucleotide molecules using a membrane channel. Proc Natl Acad Sci U S A. 1996; 93(24):13770-3. PMC: 19421. DOI: 10.1073/pnas.93.24.13770. View