Single‐Molecule Study of Peptides with the Same Amino Acid Composition but Different Sequences by Using an Aerolysin Nanopore

Hu, Fangzhou; Angelov, Borislav; Li, Shuang; Li, Na; Lin, Xubo; Zou, Aihua

doi:10.1002/cbic.202000119

Cited by 15 publications

(15 citation statements)

References 89 publications

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“…Further, when the signal to noise ratio is taken into consideration, WT AeL hardly discriminates the 20 natural amino acids according to these polymer labels with the volume exclusion model. Interestingly, previous studies revealed that short peptides with a length ranging from 6 to 14 amino acids could produce 7−10 times larger current blockage in comparison to the values estimated on the basis of the volume exclusion model 28,29 (Figure 1c and Table S5; all of the volumes of peptides were calculated from a previous study 30 ). Therefore, the simple volume exclusion model could not meet the requirements to describe the contributions of ionic current blockage.…”

mentioning

confidence: 79%

Is the Volume Exclusion Model Practicable for Nanopore Protein Sequencing?

2021

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The nanopore approach holds the possibility for achieving single-molecule protein sequencing. However, ongoing challenges still remain in the biological nanopore technology, which aims to identify 20 natural amino acids by reading the ionic current difference with the traditional current-sensing model. In this paper, taking aerolysin nanopores as an example, we calculate and compare the current blockage of each of 20 natural amino acids, which are all far from producing a detectable current blockage difference. Then, we propose a modified solution conductivity of σ′ in the traditional volume exclusion model for nanopore sensing of a peptide. The σ′ value describes the comprehensive result of ion mobility inside a nanopore, which is related to but not limited to nanopore–peptide interactions, and the positions, orientations, and conformations of peptides inside the nanopore. The nanopore experiments of a short peptide (VQIVYK) in wild type and mutant nanopores further demonstrate that the traditional volume exclusion model is not enough to fully explain the current blockage contribution and that many other factors such as enhanced nanopore–peptide interactions could contribute to a dominant part of the current change. This modified sensing model provides insights into the further development of nanopore protein sequencing methods.

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confidence: 79%

Is the Volume Exclusion Model Practicable for Nanopore Protein Sequencing?

2021

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“…Aerolysin (AeL) nanopores [24,25] have also been employed for the structural discrimination of peptides. A series of peptides with different charges and lengths [26] and with the same amino acid composition but different sequences [27] were both discriminated by using AeL nanopores. In terms of the length of peptides, it has been shown that AeL nanopores could resolve the signal of poly-arginine with a length ranging from 10 to 5 amino acids, with the resolution of one arginine [28].…”

Section: Introductionmentioning

confidence: 99%

Single polypeptide detection using a translocon EXP2 nanopore

et al. 2021

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DNA sequencing using nanopores has already been achieved and commercialized; the next step in advancing nanopore technology is towards protein sequencing. Although trials have been reported for discriminating the 20 amino acids using biological nanopores and short peptide carriers, it remains challenging. The size compatibility between nanopores and peptides is one of the issues to be addressed. Therefore, exploring biological nanopores that are suitable for peptide sensing is key in achieving amino acid sequence determination. Here, we focus on EXP2, the transmembrane protein of a translocon from malaria parasites, and describe its pore‐forming properties in the lipid bilayer. EXP2 mainly formed a nanopore with a diameter of 2.5 nm assembled from 7 monomers. Using the EXP2 nanopore allowed us to detect poly‐L‐lysine (PLL) at a single‐molecule level. Furthermore, the EXP2 nanopore has sufficient resolution to distinguish the difference in molecular weight between two individual PLL, long PLL (Mw: 30,000–70,000) and short PLL (Mw: 10,000). Our results contribute to the accumulation of information for peptide‐detectable nanopores.

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“…A complimentary direct observation technique may facilitate such interpretation. All atomistic Molecular dynamics (MD) has been used to understand conformational changes of protein inside nanopores [17][18][19] , translocation dynamics [20][21][22] , protein sensing 23 , which typically happen in a timescale of less than or of the order of 100 ns. Coarse grain models have also been used to investigate protein translocation.…”

Section: Introductionmentioning

confidence: 99%

“…Also, the interaction of the protein with the nanopore walls and the confinement effects of the nanopore may influence the motion of the protein molecule. In addition to influencing the dynamics of the protein, the confinement effect of the nanopore may cause structural changes in the protein molecule such as unfolding [17][18][19] . We will consider only the simplest case when the protein moves through the nanopore without any significant conformational changes 2 .…”

Section: Introductionmentioning

confidence: 99%

Langevin dynamics simulation of protein dynamics in nanopores at microsecond timescales

Muthukumar

Mahalik

Cifello

2021

Preprint

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With rapid advancement in the fields of nanopore analysis of protein, it has become imperative to develop modeling framework for understanding the protein dynamics in nanopores. Such modeling framework should include the effects of electro-osmosis, as it plays significant role during protein translocation in confinement. Currently, the molecular dynamics simulations that include the hydrodynamic effects are limited to a timescale of few 100 ns. These simulations give insight about important events like protein unfolding which occurs in this timescale. But many electrophoresis experiments are limited by a detector resolution of approximately 2.5 microseconds. Analytical theory has been used to interpret protein dynamics at such large timescale. There is a need for molecular modeling of more complex environment and protein shapes which cannot be accounted for by analytical theory. We have developed a framework to study globular protein dynamics in nanopores by using langevin dynamics on a rigid body model of protein and the hydrodynamics is accounted by analytical theory for simple cylindrical nanopore geometry. This framework has been applied to study the dynamics of Ubiquitin translocation in SiNx nanopore by Nir et al. They have reported 7 times decrease in average dwell time of the protein inside the nanopore in response to a small change in pH from 7.0 to 7.2 and the modification of protein charge was attributed for such drastic change. Closer examination using our simulation revealed that the electro-osmotic effects originating due to very small change in the surface electrostatic potential of the nanopore could lead to such a drastic change in protein dynamics.

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Single‐Molecule Study of Peptides with the Same Amino Acid Composition but Different Sequences by Using an Aerolysin Nanopore

Cited by 15 publications

References 89 publications

Is the Volume Exclusion Model Practicable for Nanopore Protein Sequencing?

Is the Volume Exclusion Model Practicable for Nanopore Protein Sequencing?

Single polypeptide detection using a translocon EXP2 nanopore

Langevin dynamics simulation of protein dynamics in nanopores at microsecond timescales

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