Engineering a nanopore with co-chaperonin function

Ho, Ching-Wen; Meervelt, Veerle Van; Tsai, Keng‐Chang; Temmerman, Pieter-Jan De; Mast, Jan; Maglia, Giovanni

doi:10.1126/sciadv.1500905

Cited by 39 publications

(47 citation statements)

References 63 publications

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“…Nanopore Resistive Pulse Sensing (Np-RPS) has promising applications in domains such as analytical chemistry, biotechnology and medical diagnostics123456. Np-RPS is an electrical method for the label-free detection of single molecules, which upon entry into the pore cause transient blockades of its ionic conductance.…”

mentioning

confidence: 99%

High Temperature Extends the Range of Size Discrimination of Nonionic Polymers by a Biological Nanopore

Piguet¹,

Ouldali²,

Discala³

et al. 2016

Sci Rep

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We explore the effect of temperature on the interaction of polydisperse mixtures of nonionic poly(ethylene glycol) (PEG) polymers of different average molar masses with the biological nanopore α-hemolysin. In contrast with what has been previously observed with various nanopores and analytes, we find that, for PEGs larger than a threshold molar mass (2000 g/mol, PEG 2000), increasing temperature increases the duration of the PEG/nanopore interaction. In the case of PEG 3400 the duration increases by up to a factor of 100 when the temperature increases from 5 °C to 45 °C. Importantly, we find that increasing temperature extends the polymer size range of application of nanopore-based single-molecule mass spectrometry (Np-SMMS)-type size discrimination. Indeed, in the case of PEG 3400, discrimination of individual molecular species of different monomer number is impossible at room temperature but is achieved when the temperature is raised to 45 °C. We interpret our observations as the consequence of a decrease of PEG solubility and a collapse of PEG molecules with higher temperatures. In addition to expanding the range of application of Np-SMMS to larger nonionic polymers, our findings highlight the crucial role of the polymer solubility for the nanopore detection.

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mentioning

confidence: 99%

High Temperature Extends the Range of Size Discrimination of Nonionic Polymers by a Biological Nanopore

Piguet¹,

Ouldali²,

Discala³

et al. 2016

Sci Rep

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“…This portable device is known as MinION and has been used in the analysis of nucleic acids to determine the small metagenomics of bacterial communities and to sequence viral genomes and genes from human cell lines [114][115][116][117]. Despite still possessing a higher error rate compared to that of other sequencing technologies, MinION is advantageous in that it can analyze data in real-time and in situ, ultimately making this type of technology ideal for use in the field and hospital diagnoses for the identification of pathogens [113,118,119]. In addition to nucleic acid sequencing, other areas of analysis are focused on the design of sensors based on protein nanopores, such as the identification of peptides and proteins, the monitoring of enzymatic reactions, protein folding, and glucose detection [120][121][122].…”

Section: Biosensors Based On Actinoporins Nanoporesmentioning

confidence: 99%

Actinoporins: From the Structure and Function to the Generation of Biotechnological and Therapeutic Tools

2020

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Actinoporins (APs) are a family of pore-forming toxins (PFTs) from sea anemones. These biomolecules exhibit the ability to exist as soluble monomers within an aqueous medium or as constitutively open oligomers in biological membranes. Through their conformational plasticity, actinoporins are considered good candidate molecules to be included for the rational design of molecular tools, such as immunotoxins directed against tumor cells and stochastic biosensors based on nanopores to analyze unique DNA or protein molecules. Additionally, the ability of these proteins to bind to sphingomyelin (SM) facilitates their use for the design of molecular probes to identify SM in the cells. The immunomodulatory activity of actinoporins in liposomal formulations for vaccine development has also been evaluated. In this review, we describe the potential of actinoporins for use in the development of molecular tools that could be used for possible medical and biotechnological applications.

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“…One possibility for sampling enzymatic reactions with a nanopore is to engineer an enzymatic function into the nanopore itself. In a first approach to build a catalytic nanopore, we designed an αHL nanopore with the co-catalytic function of GroES (Figure 2a-b) [49]. In bacteria, the GroEL/GroES chaperonin pair form a folding chamber of ~65 Å diameter, inside which many cellular proteins reach the native state.…”

Section: Approach 1: Engineering Nanopores With Catalytic Functionsmentioning

confidence: 99%

Single-molecule nanopore enzymology

Willems

Meervelt

Wloka

et al. 2017

Phil. Trans. R. Soc. B

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One contribution of 17 to a discussion meeting issue 'Membrane pores: from structure and assembly, to medicine and technology'. Biological nanopores are a class of membrane proteins that open nanoscale water conduits in biological membranes. When they are reconstituted in artificial membranes and a bias voltage is applied across the membrane, the ionic current passing through individual nanopores can be used to monitor chemical reactions, to recognize individual molecules and, of most interest, to sequence DNA. In addition, a more recent nanopore application is the analysis of single proteins and enzymes. Monitoring enzymatic reactions with nanopores, i.e. nanopore enzymology, has the unique advantage that it allows long-timescale observations of native proteins at the single-molecule level. Here, we describe the approaches and challenges in nanopore enzymology.This article is part of the themed issue 'Membrane pores: from structure and assembly, to medicine and technology'.

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Engineering a nanopore with co-chaperonin function

Abstract: A recombinant GroES nanopore reveals the dynamics and kinetics of the allosteric intermediates of the GroEL protein-folding reaction.

Cited by 39 publications

References 63 publications

High Temperature Extends the Range of Size Discrimination of Nonionic Polymers by a Biological Nanopore

High Temperature Extends the Range of Size Discrimination of Nonionic Polymers by a Biological Nanopore

Actinoporins: From the Structure and Function to the Generation of Biotechnological and Therapeutic Tools

Single-molecule nanopore enzymology

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