Label-Free and Real-Time Detection of Protein Ubiquitination with a Biological Nanopore

Wloka, Carsten; Meervelt, Veerle Van; Gelder, Dewi van; Danda, Natasha; Jager, Nienke; Williams, Chris P; Maglia, Giovanni

doi:10.1021/acsnano.6b07760

Cited by 116 publications

(103 citation statements)

References 49 publications

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“…Notably, the atomically precise structure of the -hemolysin protein nanopore 28 has been employed for the detection of a wide range of analytes including drugs, 29 chemical weapons, 30 nucleotides [31][32][33] as well as larger synthetic and biological supramolecular assemblies. [34][35][36] Table 1.…”

Section: Introductionmentioning

confidence: 99%

Electrostatic Forces in Field-Perturbed Equilibria: Nanopore Analysis of Cage Complexes

Borsley

Haugland

Oldknow

et al. 2019

Chem

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Molecular recognition has been widely investigated under equilibrium conditions, but little is known about such processes when perturbed by external forces. Here, we investigate the influence of electric fields on complexes formed between metallosupramolecular cages and a protein nanopore at the single-molecule level. Association rates were dominated by the applied voltage, while local electrostatic interactions between the cage and the nanopore more greatly influenced the dissociation kinetics. Exploiting these principles, it was shown that the externally applied voltage could be used to selectively bind a specific cage from a mixture containing a large excess of other cages. Moreover, the applied voltage could also be used to drive supramolecular enantio-inversion of the chiral cages, or occasionally, the nonequilibrium capture and disassembly of cages deep within the nanopore. Similar principles might be exploited in the design of other molecular devices that operate within externally applied electric fields or biogenic transmembrane potentials.

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Section: Introductionmentioning

confidence: 99%

Electrostatic Forces in Field-Perturbed Equilibria: Nanopore Analysis of Cage Complexes

Borsley

Haugland

Oldknow

et al. 2019

Chem

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“…34,35 In a typical experiment, a voltage bias is applied between two electrolyte compartments insulated by a membrane that contains a single nanopore. The bias generates a steady-state ionic current that reports on ion flow through the pore.…”

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confidence: 99%

Porphyrin-Assisted Docking of a Thermophage Portal Protein into Lipid Bilayers: Nanopore Engineering and Characterization

Cressiot¹,

Greive

et al. 2017

ACS Nano

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Nanopore-based sensors for nucleic acid sequencing and single-molecule detection typically employ pore-forming membrane proteins with hydrophobic external surfaces, suitable for insertion into a lipid bilayer. In contrast, hydrophilic pore-containing molecules, such as DNA origami, have been shown to require chemical modification to favor insertion into a lipid environment. In this work, we describe a strategy for inserting polar proteins with an inner pore into lipid membranes, focusing here on a circular 12-subunit assembly of the thermophage G20c portal protein. X-ray crystallography, electron microscopy, molecular dynamics, and thermal/chaotrope denaturation experiments all find the G20c portal protein to have a highly stable structure, favorable for nanopore sensing applications. Porphyrin conjugation to a cysteine mutant in the protein facilitates the protein’s insertion into lipid bilayers, allowing us to probe ion transport through the pore. Finally, we probed the portal interior size and shape using a series of cyclodextrins of varying sizes, revealing asymmetric transport that possibly originates from the portal’s DNA-ratchet function.

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“…Hence, it strongly influences the capture and translocation of biomolecules including nucleic acids, 36,47,122 peptides, 28,123,124 and proteins. 25,27,30,[96][97][98][99]125 Because the drag exerted by the EOF depends primarily on the size and shape of the biomolecule of interest and not on its charge, 125 it can be harnassed to capture molecules even against the electric field. 25 The EOF is a consequence of interaction between the fixed charges on the nanopore walls and mobile charges in the electrolyte.…”

Section: Transport Of Water Through Clyamentioning

confidence: 99%

“…and the solvent in terms of density, viscosity92 and relative permittivity 93 . To validate our new framework, we applied it directly to a 2D-axisymmetric model of Cytolysin A (ClyA), a large protein nanopore that typically contains 12 subunits 95 or more96 and has been extensively used in experimental studies of both proteins27,30,[96][97][98][99][100] and DNA 17,101. .…”

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confidence: 99%

Modeling of Ion and Water Transport in the Biological Nanopore ClyA

Willems

Ruić

Lucas

et al. 2020

Preprint

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In recent years, the protein nanopore cytolysin A (ClyA) has become a valuable tool for the detection, characterization and quantification of biomarkers, proteins and nucleic acids at the single-molecule level. Despite this extensive experimental utilization, a comprehensive computational study of ion and water transport through ClyA is currently lacking. Such a study yields a wealth of information on the electrolytic conditions inside the pore and on the scale the electrophoretic forces that drive molecular transport. To this end we have built a computationally efficient continuum model of ClyA which, together with an extended version of Poison-Nernst-Planck-Navier-Stokes (ePNP-NS) equations, faithfully reproduces its ionic conductance over a wide range of salt concentrations. These ePNP-NS equations aim to tackle the shortcomings of the traditional PNP-NS models by self-consistently taking into account the influence of both the ionic strength and the nanoscopic scale of the pore on all relevant electrolyte properties. In this study, we give both a detailed description of our ePNP-NS 1 model and apply it to the ClyA nanopore. This enabled us to gain a deeper insight into the influence of ionic strength and applied voltage on the ionic conductance through ClyA and a plethora of quantities difficult to assess experimentally. The latter includes the cation and anion concentrations inside the pore, the shape of the electrostatic potential landscape and the magnitude of the electro-osmotic flow. Our work shows that continuum models of biological nanopores-if the appropriate corrections are applied-can make both qualitatively and quantitatively meaningful predictions that could be valuable tool to aid in both the design and interpretation of nanopore experiments.

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Label-Free and Real-Time Detection of Protein Ubiquitination with a Biological Nanopore

Cited by 116 publications

References 49 publications

Electrostatic Forces in Field-Perturbed Equilibria: Nanopore Analysis of Cage Complexes

Electrostatic Forces in Field-Perturbed Equilibria: Nanopore Analysis of Cage Complexes

Porphyrin-Assisted Docking of a Thermophage Portal Protein into Lipid Bilayers: Nanopore Engineering and Characterization

Modeling of Ion and Water Transport in the Biological Nanopore ClyA

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