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Abstract: Colloidal quantum dots (QDs) represent a versatile class of luminescent nanomaterials whose physicochemical and interfacial properties can be engineered for advanced bio-related applications. Herein, the wet-precipitation synthesis and surface engineering of ultra-small fluorine-doped ZnO quantum dots (F/ZnO QDs) were proposed and their formulation into stable amphiphilic nanosystems using synthetic glycoglycerolipids. To control aggregation and interfacial behavior, the QDs were first capped with oleylamine and subsequently functionalized through an emulsion-based approach with mono-acyl or di-acyl glycoglycerolipids, yielding double-coated amphiphilic nanoformulations. The resulting materials were extensively characterized by TEM, DLS, zeta-potential measurements, XRD, FTIR/ATR, UV–Vis, and fluorescence spectroscopy, allowing to explore correlations between surface chemistry, colloidal stability, and optical properties. Glycoglycerolipid functionalization led to a marked improvement in aqueous dispersibility and long-term colloidal stability while preserving the enhanced fluorescence induced by fluorine doping. Biological assays confirmed the cytocompatibility of the coated QDs and supported their suitability for further biointerface studies. This work highlights glycoglycerolipid-based amphiphilic coatings as an effective strategy to tailor the surface and colloidal properties of ZnO-based QDs, enabling the development of stable luminescent nanomaterials as biocompatible nanoprobes and for bio-interfacial applications.

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Abstract: Amphiphilic copolymers have emerged as powerful, detergent-free tools for solubilizing biological membranes, enabling the extraction and stabilization of membrane proteins within native-like lipid environments. We report a comprehensive, multi-technique elucidation of how polymer:lipid stoichiometry governs the formation, size, and stability of styrene-maleic acid lipid particles (SMALPs). Using commercial SMA2000, a styrene-maleic acid copolymer made using free radical polymerization and 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC) as a model phospholipid, we prepared SMALPs across a wide range of polymer-to-lipid weight ratios and employed an integrated suite of orthogonal characterization methods, including size exclusion chromatography (SEC), dynamic light scattering (DLS), flow-induced dispersion analysis (FIDA), mass photometry, ensemble and time-resolved Förster resonance energy transfer (FRET), and small-angle X-ray scattering (SAXS), to establish the structural consequences of varying polymer content. Our data reveal that efficient lipid solubilization into nanodiscs requires a minimum amount of polymer. Above ~1% (w/v) SMA2000: 1% DMPC, well-defined nanodiscs of ~10 nm diameter are formed that, on average, exhibit a consistent stoichiometry of ~130 lipids encircled by ~11 polymer chains. Through a new molecularly-constrained SAXS model, we show that these nanodiscs possess a stable bilayer height across variations in polymer to lipid ratio and a narrow polymer belt, and that their structural parameters remain invariant once excess polymer is used. In contrast, insufficient polymer (<1% w/v) generates bimodal populations including substantially larger discs. Notably, nanodiscs formed at optimal polymer:lipid ratios remain structurally stable for at least two months. Together, these results provide a rigorous quantification of SMALP composition and preferred size, enhancing our understanding of polymer-lipid nanodisc formation and offering critical design rules for detergent-free membrane protein extraction.

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Abstract: Gram-negative bacteria pose a threat to global healthcare mainly because their outer membrane (OM) provides an intrinsic barrier to many antimicrobials. Key to this barrier function is the asymmetric structure of the OM, with phospholipids constituting the inner leaflet and lipopolysaccharides, the outer leaflet. Although the mechanism of phospholipid transport between the inner membrane (IM) and OM remains poorly understood, recent studies implicate TamB, YhdP, and YdbH as functionally redundant proteins mediating this process in Escherichia coli. Accordingly, the collective loss of these three paralogs is lethal, and any one of them is sufficient for growth. YdbH is anchored to the IM, and its periplasmic repeating β-sheet groove domain interacts with the OM lipoprotein YnbE via β-strand augmentation to form an intermembrane bridge. Additionally, YnbE multimerizes, and the periplasmic protein YdbL is proposed to modulate YnbE multimerization to facilitate its stacking on the C-terminus of YdbH. Here, we demonstrate that excess YdbL specifically inhibits the function of the YdbH-YnbE complex since overexpression of ydbL causes lethality in the ΔyhdP ΔtamB double mutant, but the presence of both ydbH and ynbE in trans abrogates this lethality. We resolve high-resolution structural data for YdbL and ascertain its interaction site with the YnbE C-terminal α-helix, with residues mediating this interface highly conserved and critical for YdbL function. Finally, we show that YdbL is protected from degradation by the protease DegP when complexed with YnbE. Overall, our data support a model in which YdbL ensures proper YdbH-YnbE intermembrane bridge formation by directly interacting with YnbE.

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Abstract: Filamin C (FLNC) is a key Z-disc protein that anchors actin filaments to membrane-bound complexes and is essential for sarcomeric stability and mechanotransduction. Genetic variants in FLNC are implicated in cardiomyopathy, a hereditary disease affecting heart muscle function. However, the structural and functional consequences of FLNC missense variants remain poorly defined, particularly those classified as variants of uncertain significance (VUS). Here, we used an integrated approach to investigate the FLNC missense variant M82K, identified in two unrelated patients presenting with dilated cardiomyopathy and associated clinical symptoms. Using structural modelling of the FLNC actin-binding domain (ABD), we predicted that the M82K variant may destabilise the ABD. Biophysical analyses of the E. coli-expressed FLNC-ABD-M82K variant displayed a ~ 10 °C decrease in melting temperature, accelerated thermolysin-mediated proteolysis, decreased solubility, and reduced actin-binding affinity. Further analysis using size exclusion chromatography coupled with and without small-angle X-ray scattering (SEC and SEC-SAXS), native PAGE, and mass photometry, all demonstrated a pronounced shift of mutant protein toward high-molecular-weight aggregates, with thermal SAXS confirming aggregation across elevated temperatures. Cellular analysis of GFP-tagged full-length FLNC-M82K showed reduced protein stability using cycloheximide. In neonatal rat cardiomyocytes there was no clear evidence of aggregation relative to wild-type in short-term transient transfections. Together, these findings support a pathogenic classification of FLNC-M82K and provide mechanistic insight into how FLNC destabilisation could lead to cardiomyopathy. More broadly, this multi-pronged strategy provides a framework for interpreting VUSs in cardiomyopathy-associated genes.

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Abstract: Chordin is a cysteine-rich protein which acts as a regulator of bone morphogenetic protein (BMP) signalling in the extracellular matrix. Acting in concert with twisted gastrulation (TWSG1), chordin works as an antagonist of BMP signalling by binding tightly to the growth factor and is a vital component of the network of interactions that establish developmental signalling gradients. Chordin is known to interact with BMP ligands via its four von-Willebrand factor type C domains, but the function of the large central four CHRD domains were previously unknown. Here we show that these domains interact strongly with sulphated glycosaminoglycans (GAGs) and provide evidence for the location of the binding site using X-ray crystallographic analysis combined with mutagenesis and biophysical techniques. Additionally, we report the first recombinant expression and purification of the complete functional chordin, TWSG1, BMP2, BMP7 complex which was used to demonstrate that the four CHRD domains are largely redundant with respect to the role of chordin as an inhibitor of BMP ligands. We therefore propose that the four CHRD domains of chordin have relevance in the diffusion and localisation of chordin-TWSG1-BMP complexes at the tissue and organismal level, mediated by their interaction with GAGs or proteoglycans.