Tuning the Size and Properties of ClyA Nanopores Assisted by Directed Evolution

Soskine, Misha; Biesemans, Annemie; Maeyer, Marc De; Maglia, Giovanni

doi:10.1021/ja4053398

Cited by 144 publications

(269 citation statements)

References 62 publications

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“…However, 5-15 min after the addition of the Aβ42 oligomers to the chamber, step-wise changes in bilayer conductance were observed ( Fig. 2A), behavior typical of the incorporation of individual proteins that form nanopores in membrane bilayers (21). As these oligomers induced various types of nanopore-like behavior, we grouped the responses into three classes, denoted type 1, 2, and 3 on the basis of the signal observed (Fig.…”

Section: The Aβ42 Oligomers Stabilized By Dpc Micelles Insert Into LImentioning

confidence: 99%

Aβ42 assembles into specific β-barrel pore-forming oligomers in membrane-mimicking environments

Serra-Batiste

Ninot-Pedrosa

Bayoumi

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The formation of amyloid-β peptide (Aβ) oligomers at the cellular membrane is considered to be a crucial process underlying neurotoxicity in Alzheimer’s disease (AD). Therefore, it is critical to characterize the oligomers that form within a membrane environment. To contribute to this characterization, we have applied strategies widely used to examine the structure of membrane proteins to study the two major Aβ variants, Aβ40 and Aβ42. Accordingly, various types of detergent micelles were extensively screened to identify one that preserved the properties of Aβ in lipid environments—namely the formation of oligomers that function as pores. Remarkably, under the optimized detergent micelle conditions, Aβ40 and Aβ42 showed different behavior. Aβ40 aggregated into amyloid fibrils, whereas Aβ42 assembled into oligomers that inserted into lipid bilayers as well-defined pores and adopted a specific structure with characteristics of a β-barrel arrangement that we named β-barrel pore-forming Aβ42 oligomers (βPFOsAβ42). Because Aβ42, relative to Aβ40, has a more prominent role in AD, the higher propensity of Aβ42 to form βPFOs constitutes an indication of their relevance in AD. Moreover, because βPFOsAβ42 adopt a specific structure, this property offers an unprecedented opportunity for testing a hypothesis regarding the involvement of βPFOs and, more generally, membrane-associated Aβ oligomers in AD.

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Section: The Aβ42 Oligomers Stabilized By Dpc Micelles Insert Into LImentioning

confidence: 99%

Aβ42 assembles into specific β-barrel pore-forming oligomers in membrane-mimicking environments

Serra-Batiste

Ninot-Pedrosa

Bayoumi

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

257

318

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“…Recently we have shown that folded proteins might be trapped transiently inside the lumen of the Cytolysin A (ClyA) nanopore without the need of complex immobilization strategies or covalent chemistry. [25][26][27][28] Remarkably, ionic current recordings could monitor the binding of ligands to internalised proteins, indicating that an enzymatic activity can be translated into an electrical signal. 28 Conveniently, ClyA could be isolated in different oligomeric states, providing nanopores with identical chemical composition but different pore dimensions.…”

Section: Introductionmentioning

confidence: 99%

“…[31][32][33][34] We have shown that ClyA nanopores can be engineered to selectively internalize proteins into the nanopore interior. [25][26][27][28] Therefore, if proteins translocate through the nanopore under physiological conditions of ionic strength and applied potential, ClyA might be used to convey therapeutic proteins across biological membranes. Proving the translocation of proteins across nanopores, however, is challenging.…”

Section: Introductionmentioning

confidence: 99%

A Protein Rotaxane Controls the Translocation of Proteins Across a ClyA Nanopore

2015

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Rotaxanes, pseudorotaxanes and catenanes are supramolecular complexes with potential use in nanomachinery, molecular computing and single-molecule studies. Here we constructed a protein rotaxane in which a polypeptide thread is encircled by a Cytolysin A (ClyA) nanopore and capped by two proteins stoppers. The rotaxane could be switched between two states. At low negative applied potentials (<−50 mV) one of the protein stoppers resided inside the nanopore indefinitely. Under this configuration the rotaxane prevents the diffusion of protein molecules across the lipid bilayer and provides a useful platform for single-molecule analysis. High negative applied potentials (−100 mV) dismantled the interlocked rotaxane system by the forceful translocation of the protein stopper, allowing new proteins to be trapped inside or transported across the nanopore. The observed voltage threshold for the translocation of the protein stopper through the nanopore related well to the biphasic voltage dependence of the residence time measured for the freely diffusing protein stopper. We propose a model in which molecules translocate through a nanopore when the average dwell time decreases with the applied potential.

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“…They also showed that these nanopores can be rendered capable of detecting selective cognate protein analytes by the attachment of appropriate aptamers at the pore mouth. In a separate study, 36 they used a directed evolution approach to tune the size and properties of ClyA nanopores. Meervelt et al 37 used the ClyA pore to investigate the interaction between the thrombin binding aptamer and human thrombin.…”

Section: Introductionmentioning

confidence: 99%

pH controlled gating of toxic protein pores by dendrimers

et al. 2016

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Designing effective nanoscale blockers for membrane inserted pores formed by pore forming toxins, which are expressed by several virulent bacterial strains, on a target cell membrane is a challenging and active area of research. Here we demonstrate that PAMAM dendrimers can act as effective pH controlled gating devices once the pore has been formed. We have used fully atomistic molecular dynamics (MD) simulations to characterize the cytolysin A (ClyA) protein pores modified with fifth generation (G5) PAMAM dendrimers. Our results show that the PAMAM dendrimer, in either its protonated (P) or non-protonated (NP) states can spontaneously enter the protein lumen. Protonated dendrimers interact strongly with the negatively charged protein pore lumen. As a consequence, P dendrimers assume a more expanded configuration efficiently blocking the pore when compared with the more compact configuration adopted by the neutral NP dendrimers creating a greater void space for the passage of water and ions. To quantify the effective blockage of the protein pore, we have calculated the pore conductance as well as the residence times by applying a weak force on the ions/water. Ionic currents are reduced by 91% for the P dendrimers and 31% for the NP dendrimers. The preferential binding of Cl(-) counter ions to the P dendrimer creates a zone of high Cl(-) concentration in the vicinity of the internalized dendrimer and a high concentration of K(+) ions in the transmembrane region of the pore lumen. In addition to steric effects, this induced charge segregation for the P dendrimer effectively blocks ionic transport through the pore. Our investigation shows that the bio-compatible PAMAM dendrimers can potentially be used to develop therapeutic protocols based on the pH sensitive gating of pores formed by pore forming toxins to mitigate bacterial infections.

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Tuning the Size and Properties of ClyA Nanopores Assisted by Directed Evolution

Cited by 144 publications

References 62 publications

Aβ42 assembles into specific β-barrel pore-forming oligomers in membrane-mimicking environments

Aβ42 assembles into specific β-barrel pore-forming oligomers in membrane-mimicking environments

A Protein Rotaxane Controls the Translocation of Proteins Across a ClyA Nanopore

pH controlled gating of toxic protein pores by dendrimers

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