Biology subjects

Maglia, G.

Publications and source records attributed to Maglia, G..

11 recordsLinked to original sources

bioRxiv2026

β-barrel nanopores designed for insertion into thick block copolymer membranes

Efficient integration of proteins into amphiphilic polymer membranes offers new opportunities in synthetic biology and nanotechnology. Long-term protein reconstitution into artificial membranes remains challenging due to a lack of stabilising protein-membrane interactions found in native lipid bilayers. Here, we redesigned the transmembrane region of a CytK-4D {beta}-barrel nanopore for stable insertion into 3.5-6.6 nm thick PBD-PEO (poly(1,2-butadiene)-b-poly(ethylene oxide)) bilayers. PBD-PEO membranes offer high mechanical and chemical stability and low electrical noise, but the thick membrane hinders anchoring of biological nanopores. By systematically investigating the elongation of the {beta}-barrel, we engineered nanopore constructs suitable for PBD11PEO8 and PBD22PEO14 membranes. Efficient insertions were observed by adding amino acids that stabilised the transmembrane {beta}-barrel structure and enhanced anchoring of the nanopore into the membrane. Molecular dynamics simulations and single-molecule assays revealed that nanopores folded naturally into PBD-PEO bilayers, enabling successful detection of cyclodextrins and translocation of polypeptides and full-length proteins. Our study offers important lessons for the reconstitution of membrane proteins into artificial membranes. Moreover, these highly robust nanopore-membrane interfaces can be readily integrated into biosensing devices, enabling peptide and protein analysis directly from complex solutions.

biophysics↗

bioRxiv2026

Single-molecule identification of full-length proteins with single-amino-acid resolution using nanopores

Nanopore-based technologies show promise in single-molecule protein sequencing. By using an unfoldase and a nanopore with enhanced electroosmotic flow, here we show the continuous identification of generic proteins during single nanopore passes. This approach enables the recording of differences in charge and size from single amino acid substitutions compared to reference signals, paving the way for single-molecule protein sequencing and high-throughput proteomics.

biophysics↗

bioRxiv2024

Assembly and dynamics of the outer membrane exopolysaccharide transporter PelBC of Pseudomonas aeruginosa

The infamous opportunistic pathogen Pseudomonas aeruginosa enhances its virulence and antibiotic resistance upon formation of durable biofilms. The biofilm stability is mediated by its matrix built of secreted exopolysaccharides, eDNA, and structural proteins. Exopolysaccharides of P. aeruginosa - Pel, Psl and alginate - have the highest biomedical relevance, but the mechanisms behind their synthesis and secretion are poorly understood. Here, we employ cryogenic electron microscopy to resolve the 2.5 [A] structure of the outer membrane complex PelBC for Pel exopolysaccharide, which is uniquely composed of the membrane-embedded {beta}-barrel PelB and the asymmetrical ring of 12 lipoproteins PelC at the periplasmic interface. The assembly captured in the lipid-based nanodisc is stabilized by electrostatic contacts of PelC with the periplasmic loops of PelB and multiple interactions with PelB N-terminal helical domains. Within the membrane, the resolved acyl chains of the PelC lipoproteins are alternated by the tryptophan residues immersed into the lipid leaflet, thus offering a stable anchoring architecture. The highly anionic interior of the PelB {beta}-barrel is sealed by three loops at the extracellular side, where the short Plug-S loop is aligned with the periplasmic helical scaffold, being the potential gating element for the Pel exopolysaccharide tunneling. Molecular dynamic simulations of PelB in native-like membrane environments suggest that Plug-S is sufficiently flexible to open a tunnel, and so serve as a gate. The gating model is further supported by single-channel conductivity measurements, which identify two conductance states of PelB. Via mutational analysis we confirm that Plug-S mediates opening of a narrow tunnel, as required for the controlled exopolysaccharide transport. Our structural and functional analysis of the pathogenicity-relevant complex offer a detailed and comprehensive view on this unique machinery and suggest the route taken by the exopolysaccharide at the final secretion step.

biophysics↗

bioRxiv2024

Hybrid lipid-block copolymer membranes enable stable reconstitution of a wide range of nanopores and robust sampling of serum

Biological nanopores are powerful tools for detecting biomolecules at the single-molecule level, making them appealing as sensors for biological samples. However, the lipid membranes in which nanopores reside can be unstable in the presence of biological fluids. Here, membranes formed with the amphiphilic polymers PMOXA-PDMS-PMOXA and PBD-PEO are tested as potential alternatives for nanopore sensing. We demonstrate that polymer membranes can possess increased stability towards applied potentials and high concentrations of human serum, but that the stable insertion of a wide range of biological nanopores is most often compromised. Alternatively, hybrid polymer-lipid membranes comprising a 1:1 w/w mixture of PBD11PEO8 and DPhPC showed high electrical and biochemical stability while creating a suitable environment for all tested nanopores. Analytes such as proteins, DNA and sugars were efficiently sampled, indicating that in hybrid membranes nanopores showed native-like properties. Molecular dynamics simulations revealed that lipids form [~]12 nm domains interspersed by a polymer matrix. Nanopores partitioned into these lipid nanodomains and sequestered lipids possibly offering the same binding strength as in a native bilayer. This work shows that single-molecule analysis with nanopores in [PBD11PEO8 + DPhPC] membranes is feasible and present stable recordings in the presence of human serum. These results pave the way towards novel nanopore-based biosensors.

biophysics↗

bioRxiv2024

Unravelled proteins form blobs during translocation across nanopores

The electroosmotic-driven transport of unravelled proteins across nanopores is an important biological process that is now under investigation for the rapid analysis and sequencing of proteins. For this approach to work, however, it is crucial that the polymer is threaded in single file. Here we found that, contrary to the electrophoretic transport of charged polymers such as DNA, during polypeptide translocation blob-like structures typically form inside nanopores. Comparisons between different nanopore sizes, shapes and surface chemistries showed that under electroosmotic-dominated regimes single-file transport of polypeptides can be achieved using nanopores that simultaneously have an entry and an internal diameter that is smaller than the persistence length of the polymer, have a uniform non-sticky (i.e. non-aromatic) nanopore inner surface, and using moderate translocation velocities.

biophysics↗

bioRxiv2023

An engineered electroosmotic flow transports unravelled proteins across nanopores

The development of a technology capable of sequencing single proteins holds promise to unravel new biological information hidden in ensemble analysis. However, new techniques must be first developed. In one approach, proteins are unfolded and translocate across a nanopore under an external bias. Unlike DNA, however, proteins do not have a uniform charge, and the electrophoretic force cannot be used to translocate proteins. Here, we show that by introducing sets of charges spaced by ~1 nm an otherwise neutral nanopore an electroosmotic force is created that induces the unidirectional transport of polypeptides, even against relatively strong electrophoretic forces. Unstructured polypeptides and native proteins unfolded with urea produce current signatures as they traversed the nanopore, which could lead to quick protein identifcation. This approach can be used to translocate and stretch proteins in non-enzymatic protein identification and enzymatic protein sequencing approaches.

biophysics↗

bioRxiv2021

Chop-n-drop: in silico assessment of a novel single-molecule protein fingerprinting method employing fragmentation and nanopore detection

The identification of proteins at the single-molecule level would open exciting new venues in biological research and disease diagnostics. Previously we proposed a nanopore-based method for protein identification called chop-n-drop fingerprinting, in which the fragmentation pattern induced and measured by a proteasome-nanopore construct is used to identify single proteins. However whether such fragmentation patterns are sufficiently characteristic of proteins to identify them in complex samples remained unclear. In the simulation study presented here, we show that 97.9% of human proteome constituents are uniquely identified under close to ideal measuring circumstances, using a simple alignment-based classification method. We show that our method is robust against experimental error, as 78.8% can still be identified if the resolution is twice as low as currently attainable and 10% of proteasome restriction sites and protein fragments are randomly ignored. Based on these results and our experimental proof-of-concept, we argue that chop-n-drop fingerprinting has the potential to make cost-effective single-molecule protein identification feasible in the near future.

bioinformatics↗

bioRxiv2020

Bottom-up fabrication of a multi-component nanopore sensor that unfolds, processes and recognizes single proteins

Transmembrane channels and pores have many biotechnological applications, notably in the single-molecule sequencing of DNA. Small synthetic nanopores have been designed using amphipathic peptides, or by assembling computationally designed transmembrane helices. The fabrication of more complex transmembrane devices has yet to be reported. In this work, we fabricated in two steps a multi-protein transmembrane device that addresses some of the main challenges in nanopore protein sequencing. In the first step, artificial nanopores are created from soluble proteins with toroid shapes. This design principle will allow fabricating a variety of nanopores for single-molecule analysis. In the second step one -subuinit of the 20S proteasome from Thermoplasma acidophilum is genetically integrated into the artificial nanopore, and a 28-component nanopore-proteasome is co-assembled in E. coli cells. This multi-component molecular machine opens the door to two new approaches in protein sequencing, in which selected substrate proteins are unfolded, fed to into the proteasomal chamber and then identified by the nanopore sensor either as intact or fragmented polypeptides. The ability to integrate molecular devices directly onto a nanopore sensors allows creating next-generation protein sequencing devices, and will shed new lights on the fundamental processes of biological nanomachines.

bioengineering↗

bioRxiv2020

Substrate binding and turnover modulate the affinity landscape of dihydrofolate reductase to increase its catalytic efficiency

It is generally accepted that enzymes structures evolved to stabilize the transition-state of a catalyzed reaction. Here, observing single molecules with a multi-turnover resolution, we provide experimental evidence for a more sophisticated narrative. We found that the binding of the NADPH cofactor to DHFR induces a first allosteric change that increases the affinity of the enzyme for the substrate. Then the enthalpy generated by the chemical step provides a power stroke that switches the enzyme to the product-bound conformations and promotes the release of the oxidized cofactor NADP+. The subsequent binding of NADPH to the vacated site provides the free energy for the recovery stroke, which induces the allosteric release of the product and resets the initial configuration. Intriguingly, the cycle is not perfect. Occasionally, DHFR undergoes second-long catalytic pauses, most likely reflecting the occupancy of an off-path conformation induced by excess energy liberated by the chemical step. This catalytic remodeling of the affinity landscape of DHFR is likely to have evolved to improve the efficiency of the reaction to cope with the high concentration of NADP+ in E. coli. And might be a general feature for complex enzymatic reaction where the binding and release of the products must be tightly controlled.

biophysics↗

bioRxiv2020

Modeling of Ion and Water Transport in the Biological Nanopore ClyA

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 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.

biophysics↗

bioRxiv2019

Aβ(1-42) tetramer and octamer structures reveal edge pores as a mechanism for membrane damage

The formation of amyloid-beta (A{beta}) oligomer pores in the membrane of neurons has been proposed as the means to explain neurotoxicity in Alzheimers disease (AD). It is therefore critical to characterize A{beta} oligomer samples in membrane-mimicking environments. Here we present the first three-dimensional structure of an A{beta} oligomer formed in dodecyl phosphocholine (DPC) micelles, namely an A{beta}(1-42) tetramer. It comprises a {beta}-sheet core made of six {beta}-strands, connected by only two {beta}-turns. The two faces of the {beta}-sheet core are hydrophobic and surrounded by the membrane-mimicking environment. In contrast, the edges of the core are hydrophilic and are solvent-exposed. By increasing the concentration of A{beta}(1-42), we prepared a sample enriched in A{beta}(1-42) octamers, formed by two A{beta}(1-42) tetramers facing each other forming a {beta}-sandwich structure. Notably, samples enriched in A{beta}(1-42) tetramers and octamers are both active in lipid bilayers and exhibit the same types of pore-like behaviour, but they show different occurrence rates. Remarkably, molecular dynamics simulations showed a new mechanism of membrane disruption in which water and ion permeation occurred through lipid-stabilized pores mediated by the hydrophilic residues located on the core {beta}-sheets edges of the A{beta}(1-42) tetramers and octamers.

biophysics↗