Transport de protéines natives, partiellement et complètement dépliées à travers des nanopores protéiques et artificiels

Nous étudions le transport de protéines natives, partiellement ou complètement dépliées à travers des nanopores protéiques ou solides à l échelle de la molécule unique en utilisant une détection électrique. Le système modèle que nous avons choisi est la protéine MalE sauvage ou mutante, en particulier la protéine MalE 219 qui se déplie à de plus faibles concentrations d agent dénaturant que la protéine sauvage. Nous montrons que la translocation de protéines partiellement dépliées à travers un canal protéique, l hémolysine du staphylocoque doré, dépend des conformations individuelles que nous pouvons distinguer. Les molécules dépliées passent rapidement dans les nanopores. Nous mesurons directement leur fraction en fonction de la concentration en agent dénaturant. La technique est très sensible aux mutations affectant le repliement.. Nous avons également étudié le transport de protéines à travers des nanopores solides dans différents cas. Nous comparons d abord le transport de protéines natives et de protéines complètement dépliées à travers un nanopore de grand diamètre puis nous étudions la translocation de protéines dépliées à travers un nanopore étroit de diamètre inférieur à la taille de la protéine. Nous observons différents régimes de translocation quand nous varions le champ électrique appliqué que nous interprétons à l aide d un modèle théorique simple.We study the transport of native, partially or completely unfolded proteins through protein or solid-state nanopores at the single molecule level using an electrical detection. The model system that we use is the wild-type MalE or mutant protein, in particular MalE219, which unfolds at lower concentration of denaturing agent than the wild type. We show that the translocation of partially unfolded proteins through the Hemolysin protein channel, a toxin from Staphylococcus aureus, depends on of individual conformations that we can distinguish. The unfolded proteins pass rapidly through the nanopores. We directly measure their proportion as a function of the concentration of denaturing agent. The technique is very sensitive to the mutations affecting the folding properties. We also study the transport of proteins through solid-state nanopores in different situations. We first compare the transport of native and fully unfolded proteins through a nanopore of large diameter. We then study the tranlocation of unfolded proteins through a narrow pore, whose diameter is smaller than the protein size. We observe different regimes of translocation by varying the applied electric field, which we interpret using a simple theoretical model.EVRY-Bib. électronique (912289901) / SudocSudocFranceF

Thermostable virus portal proteins as reprogrammable adapters for solid-state nanopore sensors

Nanopore-based sensors are advancing the sensitivity and selectivity of single-molecule detection in molecular medicine and biotechnology. Current electrical sensing devices are based on either membrane protein pores supported in planar lipid bilayers or solid-state (SS) pores fabricated in thin metallic membranes. While both types of nanosensors have been used in a variety of applications, each has inherent disadvantages that limit its use. Hybrid nanopores, consisting of a protein pore supported within a SS membrane, combine the robust nature of SS membranes with the precise and simple engineering of protein nanopores. We demonstrate here a novel lipid-free hybrid nanopore comprising a natural DNA pore from a thermostable virus, electrokinetically inserted into a larger nanopore supported in a silicon nitride membrane. The hybrid pore is stable and easy to fabricate, and, most importantly, exhibits low peripheral leakage allowing sensing and discrimination among different types of biomolecules

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

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

Transport of native, partially and completely unfolded proteins through protein and solid-state nanopores

Nous étudions le transport de protéines natives, partiellement ou complètement dépliées à travers des nanopores protéiques ou solides à l’échelle de la molécule unique en utilisant une détection électrique. Le système modèle que nous avons choisi est la protéine MalE sauvage ou mutante, en particulier la protéine MalE 219 qui se déplie à de plus faibles concentrations d’agent dénaturant que la protéine sauvage. Nous montrons que la translocation de protéines partiellement dépliées à travers un canal protéique, l’hémolysine du staphylocoque doré, dépend des conformations individuelles que nous pouvons distinguer. Les molécules dépliées passent rapidement dans les nanopores. Nous mesurons directement leur fraction en fonction de la concentration en agent dénaturant. La technique est très sensible aux mutations affectant le repliement.. Nous avons également étudié le transport de protéines à travers des nanopores solides dans différents cas. Nous comparons d’abord le transport de protéines natives et de protéines complètement dépliées à travers un nanopore de grand diamètre puis nous étudions la translocation de protéines dépliées à travers un nanopore étroit de diamètre inférieur à la taille de la protéine. Nous observons différents régimes de translocation quand nous varions le champ électrique appliqué que nous interprétons à l’aide d’un modèle théorique simple.We study the transport of native, partially or completely unfolded proteins through protein or solid-state nanopores at the single molecule level using an electrical detection. The model system that we use is the wild-type MalE or mutant protein, in particular MalE219, which unfolds at lower concentration of denaturing agent than the wild type. We show that the translocation of partially unfolded proteins through the Hemolysin protein channel, a toxin from Staphylococcus aureus, depends on of individual conformations that we can distinguish. The unfolded proteins pass rapidly through the nanopores. We directly measure their proportion as a function of the concentration of denaturing agent. The technique is very sensitive to the mutations affecting the folding properties. We also study the transport of proteins through solid-state nanopores in different situations. We first compare the transport of native and fully unfolded proteins through a nanopore of large diameter. We then study the tranlocation of unfolded proteins through a narrow pore, whose diameter is smaller than the protein size. We observe different regimes of translocation by varying the applied electric field, which we interpret using a simple theoretical model

The Promise of Nanopore Technology: Advances in the Discrimination of Protein Sequences and Chemical Modifications

International audienc

Identification of Conformational Variants for Bradykinin Biomarker Peptides from a Biofluid Using a Nanopore and Machine Learning

International audienc

Focus on Protein Unfolding Through Nanopores

International audienceIn this review, we focus only on the unfolding of proteins through nanopores. We introduce the principle of electrical detection with nanopores and how this technique provides information using an electric signal. We describe different pioneer studies on protein unfolding through protein channels and through solid-state nanopores. We discuss different methods to study protein unfolding at the single-molecule level and the advantages this new nanopore technique offers

Identification and Detection of a Peptide Biomarker and Its Enantiomer by Nanopore

International audienceUntil now, no fast, low-cost, and direct technique exists to identify and detect protein/peptide enantiomers, because their mass and charge are identical. They are essential since L- and D-protein enantiomers have different biological activities due to their unique conformations. Enantiomers have potential for diagnostic purposes for several diseases or normal bodily functions but have yet to be utilized. This work uses an aerolysin nanopore and electrical detection to identify vasopressin enantiomers, L-AVP and D-AVP, associated with different biological processes and pathologies. We show their identification according to their conformations, in either native or reducing conditions, using their specific electrical signature. To improve their identification, we used a principal component analysis approach to define the most relevant electrical parameters for their identification. Finally, we used the Monte Carlo prediction to assign each event type to a specific L- or DAVP enantiomer