Biology subjects

Dal Peraro, M.

Publications and source records attributed to Dal Peraro, M..

At least 19 recordsLinked to original sources

bioRxiv2026

The luminal and cytosolic domains of CLIMP-63 cooperate to set ER sheet width

The endoplasmic reticulum (ER) synthesized and folds secreted and transmembrane protein in regions composed of flattened sheets with a defined luminal width of [~]50 nm. CLIMP-63 has been proposed to be the principal determinant of this spacing, but the molecular mechanism by which it controls luminal width remains unclear. The luminal domain of CLIMP-63 has been proposed to act as a fixed-length spacer through antiparallel coiled-coil dimerization between molecules on opposing membranes. Here we revise this model. Using systematic cysteine trapping across the luminal domain, we demonstrate that CLIMP-63 assembles into parallel trimers. An AlphaFold-guided screen of CLIMP-63 orthologs combined with cryo-electron microscopy reveals that the luminal domain forms an elongated trimeric rod with intrinsic conformational flexibility at hinge regions. This flexibility is functionally required: replacing the human luminal domain with a more rigid ortholog causes complete ER vacuolation. Higher-order assembly of CLIMP-63 trimers beyond the trimer depends on the cytosolic tail rather than on luminal trans interactions, and the tails role in higher order assembly is separable from its other architectural functions. Together, these findings show that luminal trimeric extension and cytosolic tail-mediated clustering cooperate to determine ER sheet width.

cell biology↗

bioRxiv2026

Redox-regulated cysteine acylation governs β-lactam sensing by the Vibrio histidine kinase VbrK

The ability of pathogens to develop resistance mechanisms makes the continuous search for novel therapeutic targets indispensable. Resistance-activating systems are promising targets for restoring the efficacy of existing antibiotics. VbrK/VbrR is a two-component system from Vibrio parahaemolyticus reported as the first sensing system in gram-negatives that directly detects {beta}-lactam antibiotics. {beta}-Lactam-induced activation of this system results in the expression of the serine {beta}-lactamase CARB. In this study, we provide insights into the mechanism of {beta}-lactam binding to the periplasmic sensor domain of the histidine kinase VbrK. Our results demonstrate that the interaction depends on the redox state of cysteines C86 and C107, highlighting the role of disulfide bond dynamics in modulating ligand recognition. We further show that formation of a non-covalent complex leads to acylation of the sensor domain by {beta}-lactams, a modification that is slowly reversed through de-acylation, yielding the hydrolyzed {beta}-lactam ring and allowing for recovery from induction once the antibiotic has been depleted from the environment. Together, these findings reveal a previously unrecognized redox- and covalent chemistry-dependent mode of {beta}-lactam interaction with histidine kinases, providing a molecular framework to understand how VbrK detects and responds to {beta}-lactam antibiotics, and opening new avenues to prevent manifestation of resistance. Short broader audience statementBacterial pathogens can sense the presence of antibiotics and trigger resistance responses, making infections increasingly difficult to treat. In this study, we uncover a previously unknown mechanism by which {beta}-lactam antibiotics interact with a sensor protein that activates resistance in Vibrio parahaemolyticus. These findings pave the way for designing new compounds that can block this interaction and thus restore the effectiveness of {beta}-lactam antibiotics against gastrointestinal infections caused by resistant bacteria.

microbiology↗

bioRxiv2025

Sub-Nanomolar Detection and Discrimination of Microcystin Congeners Using Aerolysin Nanopores

Climate-driven disruptions in aquatic ecosystems are amplifying cyanotoxin production, threatening drinking and recreational water safety. Monitoring of these toxins is challenged by requirements of the low {micro}g/L detections limits and structural diversity. Here, we employ aerolysin nanopores to distinguish seven of the most prevalent microcystin congeners, both individually and in mixtures, at environmentally relevant concentrations. Importantly, we showed that aerolysin enables the detection of microcystins in different lake water samples at concentrations below the World Health Organizations intervention thresholds, reaching picomolar sensitivity. Moreover, combining experiments and molecular dynamics simulations, we further investigated the microcystin sensing mechanism, suggesting that the ionic current blockage is primarily governed by K238 in aerolysin, while dwell time is regulated by R220 constriction site. Our results open the way to use the nanopore sensing technology for real-time monitoring of microcystins in drinking water sources and surface waters.

bioengineering↗

bioRxiv2025

Molecular Regulation and Physiological Role of 1 GOLPH3-mediated Golgi retention

The Golgi complex serves as the central hub of the biosynthetic pathway, where anterograde and retrograde trafficking converge. How cargo and Golgi-resident proteins traverse this organelle has long been debated. Recent studies have identified a molecular machinery that sorts resident proteins into retrograde-directed COPI vesicles during cisternal maturation. Golgi phosphoprotein 3 (GOLPH3) is a key component of this system; however, its physiological relevance and regulatory mechanisms remain poorly defined. Here, we show that GOLPH3 depletion in mice disrupts both protein and lipid glycosylation, causes partially penetrant embryonic lethality, and severely impairs growth and bone mineralization. At the molecular level, we find that GOLPH3 is regulated by functionally antagonistic S-acylation events that control the topology of its membrane association. To mediate retrograde trafficking of Golgi-resident glycosyltransferases, GOLPH3 must bind their cytosolic tails. This occurs via a negatively charged surface region, which is correctly oriented only in one of the S-acylated GOLPH3 conformations. Together, these findings reveal a lipid-mediated regulatory mechanism for intra-Golgi trafficking and establish the critical role of GOLPH3 in vertebrate development.

cell biology↗

bioRxiv2025

Context-aware geometric deep learning for RNA sequence design

RNA design has emerged to play a crucial role in synthetic biology and therapeutics. Although tertiary structure-based RNA design methods have been developed recently, they still overlook the broader molecular context, such as interactions with proteins, ligands, DNA, or ions, limiting the accuracy and functionality of designed sequences. To address this challenge, we present RISoTTo (RIbonucleic acid Sequence design from TerTiary structure), a parameter-free geometric deep learning approach that generates RNA sequences conditioned on both their backbone scaffolds and the surrounding molecular context. We evaluate the designed sequences based on their native sequence recovery rate and further validate them by predicting their secondary structures in silico and comparing them to the corresponding native structures. RISoTTo performs well on both metrics, demonstrating its ability to generate accurate and structurally consistent RNA sequences. Additionally, we present an in silico design study of domain 1 of the NAD+ riboswitch, where RISoTTo-generated sequences are predicted to exhibit enhanced binding affinity for both the U1A protein and the NAD+ ligand.

bioinformatics↗

bioRxiv2024

OXA beta-lactamases from Acinetobacter spp. are membrane-bound and secreted into outer membrane vesicles

{beta}-lactamases from Gram-negative bacteria are generally regarded as soluble, periplasmic enzymes. NDMs have been exceptionally characterized as lipoproteins anchored to the outer membrane. A bioinformatics study on all sequenced {beta}-lactamases was performed that revealed a predominance of putative lipidated enzymes in the class D OXAs. Namely, 60% of the OXA class D enzymes contain a lipobox sequence in their signal peptide, that is expected to trigger lipidation and membrane anchoring. This contrasts with {beta}-lactamases from other classes, which are predicted to be mostly soluble proteins. Almost all (> 99%) putative lipidated OXAs are present in Acinetobacter spp. Importantly, we further demonstrate that OXA-23 and OXA-24/40 are lipidated, membrane-bound proteins in Acinetobacter baumannii. In contrast, OXA-48 (commonly produced by Enterobacterales) lacks a lipobox and is a soluble protein. Outer membrane vesicles (OMVs) from Acinetobacter baumannii cells expressing OXA-23 and OXA-24/40 contain these enzymes in their active form. Moreover, OXA-loaded OMVs were able to protect A. baumannii, Escherichia coli and Pseudomonas aeruginosa cells susceptible to piperacillin and imipenem. These results permit us to conclude that membrane binding is a bacterial host-specific phenomenon in OXA enzymes. These findings reveal that membrane-bound {beta}-lactamases are more common than expected and support the hypothesis that OMVs loaded with lipidated {beta}-lactamases are vehicles for antimicrobial resistance and its dissemination. This advantage could be crucial in polymicrobial infections, in which Acinetobacter spp. are usually involved, and underscore the relevance of identifying the cellular localization of lactamases to better understand their physiology and target them. IMPORTANCE{beta}-lactamases represent the main mechanism of antimicrobial resistance in Gram-negative pathogens. Their catalytic function (cleaving {beta}-lactam antibiotics) occurs in the bacterial periplasm, where they are commonly reported as soluble proteins. A bioinformatic analysis reveals a significant number of putative lipidated {beta}-lactamases, expected to be attached to the outer bacterial membrane. Notably, 60% of class D OXA {beta}-lactamases (all from Acinetobacter spp) are predicted as membrane-anchored proteins. We demonstrate that two clinically relevant carbapenemases, OXA-23 and OXA-24/40 are membrane-bound proteins in A. baumannii. This cellular localization favors secretion of these enzymes into outer membrane vesicles that transport them outside the boundaries of the cell. {beta}-lactamase-loaded vesicles can protect populations of antibiotic-susceptible bacteria, enabling them to thrive in the presence of {beta}-lactam antibiotics. The ubiquity of this phenomenon suggests that it may have influenced the dissemination of resistance mediated by Acinetobacter spp., particularly in polymicrobial infections, being a potent evolutionary advantage.

microbiology↗

bioRxiv2024

Lumen charge governs ion transport in β-barrel nanopores

{beta}-barrel nanopores are involved in crucial biological processes, from ATP export in mitochondria to bacterial resistance, and represent a promising platform for emerging sequencing technologies. However, in contrast to ion channels, the understanding of the fundamental principles governing ion transport through these nanopores remains in its early stages. In this study, we integrate experimental, numerical, and theoretical approaches to elucidate ion transport mechanisms in these biological nanopores. We identify and characterise two distinct nonlinear phenomena: open-pore rectification and gating. Through extensive mutation analysis of aerolysin nanopores, we demonstrate that open-pore rectification is caused by ionic accumulation driven by the distribution of lumen charges. Additionally, we provide converging evidence suggesting that gating is controlled by electric fields dissociating counterions from lumen charges, promoting local structural deformations. Our findings establish a rigorous framework for the characterisation and understanding of biological ion transport processes, enabling the design of adaptable biosensors. We illustrate this by optimizing an aerolysin mutant for computing applications, paving the way for novel nanofluidic technologies.

biophysics↗

bioRxiv2024

Multiplexed lifetime imaging of single molecules with a gated single-photon camera

Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to discriminate fluorescent molecules or probe their nanoscale environment. Traditionally, FLIM uses time-correlated single-photon counting (TCSPC), which is precise but intrinsically low-throughput due to its dependence on point detectors. Although time-gated cameras have demonstrated the potential for high-throughput FLIM in bright samples with dense labeling, their use in single-molecule microscopy has not been explored extensively. Here, we report fast and accurate single-molecule FLIM with a commercial time-gated single-photon camera. Our optimized acquisition scheme achieves single-molecule lifetime measurements with a precision only about three times less than TCSPC, while imaging with a large number of pixels (512x512) allowing for the spatial multiplexing of over 3000 molecules. With this approach, we demonstrate parallelized lifetime measurements of large numbers of labeled pore-forming proteins on supported lipid bilayers, and temporal single-molecule Forster resonance energy transfer measurements at 5-25 Hz. This method holds considerable promise for the advancement of multi-target single-molecule localization microscopy and biopolymer sequencing.

biophysics↗

bioRxiv2024

Aerolysin nanopore structure revealed at high resolution in lipid environment

Aerolysin is a {beta}-pore-forming toxin produced by most Aeromonas bacteria which has attracted large attention in the field of nanopore sensing due to its narrow and charged pore lumen. Structurally similar proteins, belonging to the aerolysin-like family, are present throughout all kingdoms of life, but very few of them have been structurally characterized in a lipid environment. Here we present the first high-resolution atomic cryo-EM structures of aerolysin pre-pore and pore in a membrane-like environment. These structures allow the identification of key interactions, which are relevant for the pore formation and for positioning the pore barrel into the membrane with the anchoring {beta}-turn motif now finally observed. Moreover, we elucidate at high resolution the architecture of key mutations and precisely identify four constriction rings in the pore lumen that are highly relevant for nanopore sensing experiments.

biochemistry↗

bioRxiv2024

Cardiolipin clustering promotes mitochondrial membrane dynamics

Cardiolipin (CL) is a mitochondria-specific phospholipid that forms heterotypic interactions with membrane-shaping proteins and regulates the dynamic remodeling and function of mitochondria. However, the precise mechanisms through which CL influences mitochondrial morphology are not well understood. In this study, employing molecular dynamics (MD) simulations, we determined that CL molecules extensively engage with the paddle domain (PD) of mitochondrial fusion protein Optic Atrophy 1 (OPA1), which controls membrane-shaping mechanisms. Structure-function analysis confirmed the interactions between CL and two conserved motifs of OPA1 at the membrane-binding sites. We further developed a bromine-labeled CL probe to enhance cryoEM contrast and characterized the structure of OPA1 assemblies bound to the CL-brominated lipid bilayers. Our images provide direct evidence of CL enrichment within the OPA1-binding leaflet. Last, we observed a decrease in membrane remodeling activity for OPA1 in lipid compositions with increasing concentrations of monolyso-cardiolipin (MLCL). This suggests that the partial replacement of CL by MLCL, as observed in Barth syndrome-associated mutations of the tafazzin phospholipid transacylase, alters the malleability of the membrane and compromises proper remodeling. Together, these data provide insights into how biological membranes regulate the mechanisms governing mitochondrial homeostasis.

biophysics↗

bioRxiv2024

A look beyond the QR code of SNARE proteins

Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor (SNARE) proteins catalyze the fusion process of vesicles with target membranes in eukaryotic cells. To do this, they assemble in a zipper-like fashion into stable complexes between the membranes. Structural studies have shown that the complexes consist of four different helices, which we subdivide into Qa-, Qb-, Qc-, and R-helix on the basis of their sequence signatures. Using a combination of biochemistry, modeling and molecular dynamics, we investigated how the four different types are arranged in a complex. We found that there is a matching pattern in the core of the complex that dictates the position of the four fundamental SNARE types in the bundle, resulting in a QabcR complex. In the cell, several different cognate QabcR-SNARE complexes catalyze the different transport steps between the compartments of the endomembrane system. Each of these cognate QabcR complexes is compiled from a repertoire of about 20 SNARE subtypes. Our studies show that exchange within the four types is largely tolerated structurally, although some non-cognate exchanges lead to structural imbalances. This suggests that SNARE complexes have evolved for a catalytic mechanism, a mechanism that leaves little scope for selectivity beyond the QabcR rule.

biochemistry↗

bioRxiv2024

Exploring new nanopore sensors from the aerolysin family

Aerolysin-like proteins are a family of {beta}-pore-forming toxins which are widely present in all kingdoms of life. Recently, this family of proteins is gaining attention because of their biotechnological application as nanopore sensors for sensing and sequencing of biomolecules. Here, we explore the possibilities of using the knowledge of the sequence and structure of proteins to screen and explore new potential nanopore candidates. However, in spite of the conserved structural fold, the sequence identity in this family is very low. This complicates their sequence alignment, hindering the understanding of their pore structure and properties, therefore limiting further biotechnological applications. In an attempt to further understand the properties of aerolysin-like pores, we analyzed the pore structure of three family members, Clostridium perfringens epsilon toxin (ETX), Laetiporus sulphureus lectin (LSL) and Bacillus thuringiensis parasporin-2, comparing it to aerolysin. Their structure and sensing capabilities for ssDNA were first assessed by in silico methods. Moreover, ETX was characterized experimentally in planar lipid membranes for the detection of biomolecules. We found that ETX can form three distinct pore conformations, each presenting a specific open pore current, and only one of them being able to translocate ssDNA. When the ssDNA translocate through ETX, the depth of current blockage is higher compared to aerolysin which indicates a higher sensitivity for molecular sensing. Our findings open a new venue for improving and diversifying nanopore capabilities for molecular sensing.

biophysics↗

bioRxiv2024

Protein language models learn evolutionary statistics of interacting sequence motifs

Protein language models (pLMs) have emerged as potent tools for predicting and designing protein structure and function, and the degree to which these models fundamentally understand the inherent biophysics of protein structure stands as an open question. Motivated by a discovery that pLM-based structure predictors erroneously predict nonphysical structures for protein isoforms, we investigated the nature of sequence context needed for contact predictions in the pLM ESM-2. We demonstrate by use of a "categorical Jacobian" calculation that ESM-2 stores statistics of coevolving residues, analogously to simpler modelling approaches like Markov Random Fields and Multivariate Gaussian models. We further investigated how ESM-2 "stores" information needed to predict contacts by comparing sequence masking strategies, and found that providing local windows of sequence information allowed ESM-2 to best recover predicted contacts. This suggests that pLMs predict contacts by storing motifs of pairwise contacts. Our investigation highlights the limitations of current pLMs and underscores the importance of understanding the underlying mechanisms of these models. Significance StatementProtein language models (pLMs) have exhibited remarkable capabilities in protein structure prediction and design. However, the extent to which they comprehend the intrinsic biophysics of protein structures remains uncertain. We present a suite of analyses that dissect how the flagship pLM ESM-2 predicts structure. Motivated by a consistent error of protein isoforms predicted as structured fragments, we developed a completely unsupervised method to uniformly evaluate any protein language model that allows for us to compare coevolutionary statistics to older linear models. We further identified t hat E SM-2 a ppears to have a precise context size that is needed to predict inter-residue contacts. Our study highlights the current limitations of pLMs and contributes to a deeper understanding of their underlying mechanisms, paving the way for more reliable protein structure predictions.

bioinformatics↗

bioRxiv2024

Dissecting the membrane association mechanism of aerolysin pore at femtomolar concentrations using water as a probe

Aerolysin is a bacterial pore-forming toxin able to form transmembrane pores at the host plasma membrane of narrow internal diameter and great stability. These assets make it a highly promising nanopore for the detection of biopolymers such as nucleic acids and peptides. While much is known about aerolysin from a microbiological and structural side, its membrane association and pore-formation mechanism are not yet fully disclosed. Here, we studied the interaction of femtomolar concentrations of aerolysin and its mutants with liposomes in aqueous solution using angle-resolved second harmonic scattering (AR-SHS), in combination with single-channel current measurements. The measurements were so sensitive to detect electrostatic changes on the membrane-bound aerolysin induced by pH variation induced by the changes in the hydration shell of aerolysin. We reported for the first time the membrane binding affinity of aerolysin at different stages of the pore formation mechanism: while wt aerolysin has a binding affinity as high as 20 fM, the quasi-pore state and the prepore state show gradually decreasing membrane affinities, incomplete insertion and pore opening signature. Moreover, we quantitatively characterized the membrane affinity of mutants relevant for applications to nanopore sensing. This approach opens new possibilities to efficiently screen biological pores suitable for conducting molecular sensing and sequencing measurements, as well as to probe pore forming processes.

biophysics↗

bioRxiv2023

Missense mutations in CMS22 patients reveal that PREPL has both enzymatic and non-enzymatic functions

Congenital myasthenic syndrome-22 (CMS22) is a rare genetic disorder caused by mutations in the prolyl endopeptidase-like (PREPL) gene. Since previous reports only described patients with deletions and nonsense mutations in PREPL, nothing is known about the effect of missense mutations in the pathology of CMS22. In this study, we have characterized missense mutations in PREPL in three CMS22 patients, all with hallmark phenotypes. Biochemical evaluation revealed that these missense mutations do not impair hydrolase activity, thereby challenging the conventional diagnostic criteria. Structural analysis shows that the mutations affect regions most likely involved in intra-protein or protein-protein interactions. Indeed, binding to a selected group of known interactors was differentially reduced for the three mutants. The importance of non-hydrolytic functions of PREPL was investigated in catalytically inactive PREPL p.Ser559Ala cell lines which showed that hydrolytic activity of PREPL is needed for normal mitochondrial function but not for regulating AP1-mediated transport in the trans-Golgi network. In conclusion, these studies show that CMS22 can be caused not only by deletion and truncation of PREPL but also by missense mutations that do not necessarily result in a loss of hydrolytic activity of PREPL. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/572145v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@13dc0bborg.highwire.dtl.DTLVardef@13865d6org.highwire.dtl.DTLVardef@1a5ed3borg.highwire.dtl.DTLVardef@79fdf5_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2023

Differential Effects of Posttranslational Modifications on the Membrane Interaction of Huntingtin Protein

Huntingtons disease (HD) is a neurodegenerative disorder caused by an expanded polyglutamine stretch near the N-terminus of the huntingtin (HTT) protein, rendering the protein more prone to aggregate. The first 17 residues in HTT (Nt17) interact with lipid membranes and harbor multiple posttranslational modifications (PTMs) that can modulate HTT conformation and aggregation. In this study, we used a combination of biophysical studies and molecular simulations to investigate the impact of PTMs on the helicity of Nt17 in the presence of various lipid membranes. We demonstrate that anionic lipids such as PI4P, PI(4,5)P2, and GM1 significantly enhance the helical structure of unmodified Nt17. This effect is attenuated by single acetylation events at K6, K9, or K15, whereas tri-acetylation at these sites abolishes Nt17 membrane interaction. Similarly, single phosphorylation at S13 and S16 decreased but did not abolish the POPG and PIP2-induced helicity, while dual phosphorylation at these sites markedly diminished Nt17 helicity, regardless of lipid composition. The helicity of Nt17 with phosphorylation at T3 is insensitive to the membrane environment. Oxidation at M8 variably affects membrane-induced helicity, highlighting a lipid-dependent modulation of Nt17 structure. Altogether, our findings reveal differential effects of PTMs and crosstalks between PTMs on membrane interaction and conformation of HTT. Intriguingly, the effects of phosphorylation at T3 or single acetylation at K6, K9, and K15 on Nt17 conformation in the presence of certain membranes do not mirror that observed in the absence of membranes. Our studies provide novel insight into the complex relationship between Nt17 structure, PTMs and membrane binding. TOC figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/569526v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@153610eorg.highwire.dtl.DTLVardef@10908dforg.highwire.dtl.DTLVardef@256c14org.highwire.dtl.DTLVardef@dac763_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

MolecularWebXR: Multiuser discussions about chemistry and biology in immersive and inclusive VR

MolecularWebXR is a new website for education, science communication and scientific peer discussion in chemistry and biology, based on modern web-based Virtual Reality (VR) and Augmented Reality (AR). With no installs as it is all web-served, MolecularWebXR enables multiple users to simultaneously explore, communicate and discuss concepts about chemistry and biology in immersive 3D environments, by manipulating and passing around objects with their bare hands and pointing at different elements with natural hand gestures. User may either be present in the same real space or distributed around the world, in the latter case talking naturally with each other thanks to built-in audio features. Although MolecularWebXR is most immersive when running in the web browsers of high-end AR/VR headsets, its WebXR core also allows participation by users with consumer devices such as smartphones, possibly inserted into cardboard goggles for deeper immersivity, or even in computers and tablets. MolecularWebXR comes with preset VR rooms that cover topics from general, inorganic and organic chemistry, biophysics and structural biology, and general biology; besides, new content can be added at will through moleculARwebs PDB2AR tool or by contacting the lead authors. We verified MolecularWebXRs ease of use and versatility by people aged 12-80 years old in entirely virtual sessions or in mixed real-virtual sessions at various science outreach events, in courses at the bachelor, masters and early doctoral levels, in scientific collaborations, and in conference lectures. MolecularWebXR is available for free use without registration at https://molecularwebxr.org, and a blog post version of this preprint with embedded videos is available at https://go.epfl.ch/molecularwebxr-blog-post.

scientific communication and education↗

bioRxiv2023

Deep learning-assisted single-molecule detection of protein post-translational modifications with a biological nanopore

Protein post-translational modifications (PTMs) play a crucial role in countless biological processes, profoundly modulating protein properties on both the spatial and temporal scales. Protein PTMs have also emerged as reliable biomarkers for several diseases. However, only a handful of techniques are available to accurately measure their levels, capture their complexity at a single molecule level and characterize their multifaceted roles in health and disease. Nanopore sensing provides high sensitivity for the detection of low-abundance proteins, holding the potential to impact single-molecule proteomics and PTM detection in particular. Here, we demonstrate the ability of a biological nanopore, the pore-forming toxin aerolysin, to detect and distinguish -synuclein-derived peptides bearing single or multiple PTMs, namely phosphorylation, nitration and oxidation occurring at different positions and in various combinations. The characteristic current signatures of the -synuclein peptide and its PTM variants could be confidently identified using a deep learning model for signal processing. We further demonstrate that this framework can quantify -synuclein peptides at picomolar concentration and detect the C-terminal peptides generated by digestion of full-length -synuclein. Collectively, our work highlights the unique advantage of using nanopore as a tool for simultaneous detection of multiple PTMs and paves the way for their use in biomarker discovery and diagnostics.

biophysics↗