Control of subunit stoichiometry in single-chain MspA nanopores

Pavlenok, Mikhail; Yu, Luning; Herrmann, Dominik; Wanunu, Meni; Niederweis, Michael

doi:10.1016/j.bpj.2022.01.022

Cited by 11 publications

(19 citation statements)

References 77 publications

(81 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We envision the use of this effect in future nanopore-based single-molecule protein sequencing applications. For example, high-bandwidth measurements can be combined with GdmCl-mediated protein transport through a higher-resolution pore (such as MspA, which maintains its functionality in 2 M GdmCl 57 ) or asymmetric buffer conditions in which protein unfolding is mediated in one chamber, electroosmotic forces are used to keep the protein taut at the pore, and enzyme-mediated motion on another chamber of the pore is used to move the protein through the pore in a discrete manner. Moreover, recent work has shown that the use of different anions (such as nitrate) in the buffer has an effect of reducing the noise as compared to chloride 58 , which could in principle be used to further improve the signal.…”

Section: Discussionmentioning

confidence: 99%

Unidirectional single-file transport of full-length proteins through a nanopore

Kang

et al. 2023

Nat Biotechnol

Self Cite

View full text Add to dashboard Cite

Nanopore sequencing is one of only a few methods that can potentially determine the amino acid sequence of individual protein molecules as these are passed through a pore sensor. However, mechanisms for unfolding and translocation of proteins are still unavailable to date. Here we describe a general approach for realizing unidirectional transport of full-length proteins through nanopores. We combine a chemically resistant biological nanopore platform with a high concentration guanidinium chloride buffer to achieve unidirectional, single-file protein transport that is propelled by a giant electro-osmotic effect, as revealed by molecular dynamics simulations and confirmed experimentally. Remarkably, we observed that protein velocities are uniform regardless of the protein sequence, which allows the identification and discrimination among proteins based on their electrical signatures, as well as to distinguish protein signatures by their threading orientation (N-to-C vs. C-to-N terminus). With average transport velocities of 10 µs per amino acid, our method can enable direct, enzyme-free protein fingerprinting and protein sequencing when combined with a high-resolution pore and high-speed nanopore readout.

show abstract

Section: Discussionmentioning

confidence: 99%

Unidirectional single-file transport of full-length proteins through a nanopore

Kang

et al. 2023

Nat Biotechnol

Self Cite

View full text Add to dashboard Cite

show abstract

“…We tested CRP (125 kDa), an important inflammatory biomarker whose concentration in blood is related to sepsis, which is one of the main causes of death in patients in intensive care units. , Although the fragility of the lipid bilayer did not allow us to sample real biological samples, we showed here that CRP could be discriminated at clinically relevant concentrations (>2.5 mg/L), and that the presence of other human proteins in the sample seemed not to alter the CRP signature. Hence, if the stability of the lipid bilayer can be improved, for instance by using synthetic polymer membrane, , the nanopore approach might be used in the clinic or at home for the real-time and continuous monitoring of protein biomarkers.…”

Section: Discussionmentioning

confidence: 99%

Protein Sizing with 15 nm Conical Biological Nanopore YaxAB

et al. 2023

View full text Add to dashboard Cite

Nanopores are promising single-molecule tools for the electrical identification and sequencing of biomolecules. However, the characterization of proteins, especially in real-time and in complex biological samples, is complicated by the sheer variety of sizes and shapes in the proteome. Here, we introduce a large biological nanopore, YaxAB for folded protein analysis. The 15 nm cis-opening and a 3.5 nm trans-constriction describe a conical shape that allows the characterization of a wide range of proteins. Molecular dynamics showed proteins are captured by the electroosmotic flow, and the overall resistance is largely dominated by the narrow trans constriction region of the nanopore. Conveniently, proteins in the 35−125 kDa range remain trapped within the conical lumen of the nanopore for a time that can be tuned by the external bias. Contrary to cylindrical nanopores, in YaxAB, the current blockade decreases with the size of the trapped protein, as smaller proteins penetrate deeper into the constriction region than larger proteins do. These characteristics are especially useful for characterizing large proteins, as shown for pentameric C-reactive protein (125 kDa), a widely used health indicator, which showed a signal that could be identified in the background of other serum proteins.

show abstract

“…299 Other than a nanopore's macroscopic morphology, advanced method-based functionalization of the nanopore's inner surfaces can also be implemented. For example, chemical modification, 300−303 mutations, 176,304,305 and combination with aptamer molecules, 306,307 etc., have the potential to provide the required sensitivity, selectivity, and capture efficiency by manipulating interactions between proteins/peptides/AAs and nanopores. 308−310 All of these advances will be complemented by improvements in proteinfolding predictions.…”

Section: Challenges and Opportunitiesmentioning

confidence: 99%

“…173−175 The homo-octameric "globlet-like" configuration of the Mycobacterium smegmatis porin A (MspA) nanopore features a ∼1.2 nm (in diameter) constriction located at the very bottom of the channel, which could procure better read lengths of some analytes and particularly high current levels, relative to other protein pores. 176 Bai et al selected the MspA nanopore for its unique geometry to test the idea of conjugating a target peptide to a single-stranded DNA handle, regulated by a DNA helicase, to better control nanopore-based peptide transport through the pore (Figure 4a). 177 Their method was able to thread peptides of up to 17 AAs through the constriction of the MspA pore and differentiate AA residues based on their charge and position.…”

Section: Manipulating the Motion Of Peptidesmentioning

confidence: 99%

“…Other than a nanopore’s macroscopic morphology, advanced method-based functionalization of the nanopore’s inner surfaces can also be implemented. For example, chemical modification, − mutations, ,, and combination with aptamer molecules, , etc., have the potential to provide the required sensitivity, selectivity, and capture efficiency by manipulating interactions between proteins/peptides/AAs and nanopores. − All of these advances will be complemented by improvements in protein-folding predictions. From the current recording and signal analysis perspective, some noteworthy investigations have been performed on current noise fluctuations and signal bandwidth, ,, calibration of instrument differences, improving signal-to-noise ratio (SNR), increasing signal specificity, , and introducing more dimensional parameters for data analysis, etc., which have made significant strides in the nanobiotechnology arena.…”

Section: Challenges and Opportunitiesmentioning

confidence: 99%

See 1 more Smart Citation

Engineering Biological Nanopore Approaches toward Protein Sequencing

Wei,

Penkauskas,

Reiner

et al. 2023

ACS Nano

View full text Add to dashboard Cite

Biotechnological innovations have vastly improved the capacity to perform large-scale protein studies, while the methods we have for identifying and quantifying individual proteins are still inadequate to perform protein sequencing at the single-molecule level. Nanopore-inspired systems devoted to understanding how single molecules behave have been extensively developed for applications in genome sequencing. These nanopore systems are emerging as prominent tools for protein identification, detection, and analysis, suggesting realistic prospects for novel protein sequencing. This review summarizes recent advances in biological nanopore sensors toward protein sequencing, from the identification of individual amino acids to the controlled translocation of peptides and proteins, with attention focused on device and algorithm development and the delineation of molecular mechanisms with the aid of simulations. Specifically, the review aims to offer recommendations for the advancement of nanopore-based protein sequencing from an engineering perspective, highlighting the need for collaborative efforts across multiple disciplines. These efforts should include chemical conjugation, protein engineering, molecular simulation, machine-learning-assisted identification, and electronic device fabrication to enable practical implementation in real-world scenarios.

show abstract

Control of subunit stoichiometry in single-chain MspA nanopores

Cited by 11 publications

References 77 publications

Unidirectional single-file transport of full-length proteins through a nanopore

Unidirectional single-file transport of full-length proteins through a nanopore

Protein Sizing with 15 nm Conical Biological Nanopore YaxAB

Engineering Biological Nanopore Approaches toward Protein Sequencing

Contact Info

Product

Resources

About