Biochimica et Biophysica Acta (BBA) - Biomembranes

Small-angle X-ray or neutron scattering (SAXS/SANS) analysis of large unilamellar vesicles (LUVs) is often limited by high-dimensional bilayer models and the lack of dedicated, statistically rigorous workflows. Here, we introduce SAS_MoCa, an open-source Python package that integrates a compositional scattering density profile (SDP) description of lipid bilayers with a separated form factor (SFF) treatment of vesicle size and polydispersity, and couples these highly parameterized models to an adaptive thermodynamic simulated annealing algorithm formulated within a constrained Bayesian framework. SAS_MoCa enables users to incorporate quantitative prior information from, e.g., previous SAXS/SANS studies, dynamic light scattering, NMR, or molecular simulations, and returns full posterior parameter distributions, uncertainties (reported as medians and median absolute deviations) and correlations even from single SAXS curves. Validation on POPC, POPE and DMPC SAXS-only data demonstrates that the method yields reproducible structural parameters with uncertainties comparable to joint SAXS/contrast-variation SANS analyses. The modular architecture of SAS_MoCa facilitates extension to additional lipid systems and future joint SAXS/SANS or SANS-only applications.

We present a comparative study of the inner membrane of three Gram-positive bacterial strains, namely S. aureus, S. epidermidis and N. lacusekhoensis. A lipidomics study is used to obtain the lipid architecture and composition for S. epidermidis found in the skin microbiome and N. lacusekhoensis, an extremophile present in halophilic and alkophilic environments. Differences between the strains arise from both the lipid architecture and the cardiolipin content varying from 5% in S. aureus to 85% in N. lacusekhoensis. We develop coarse grained (CG) Martini-3 membrane models which reproduce structural properties such as membrane area, thickness, density distributions as well as ion-correlations with all-atom models. Inter-lipid correlations reveal a homogeneous distribution of lipids in the membranes despite the wide variation in lipid types and composition. Mechanical properties such as the area stretch modulus increased with cardiolipin content, however the bending modulus has a more complex dependence on membrane charge and lipid type. Using the CG models we evaluate the insertion free energies for four widely used antimicrobial molecules. Entry barriers for thymol and methylparaben arise from the charge density modulation at the membrane headgroups due to counterion condensation. The entry mechanisms of the antimicrobial peptide cecropin-melittin-15 (CM15) and the preservative molecule ethyl-lauroyl-arginate (ELAR) are found to be similar across all three strains. We also illustrate the manner in which the extremophilic strain, N. lacusekhoensis with its high cardiolipin content, modulates the partitioning kinetics of the antimicrobial molecule thymol with pH and salt. Our study reveals that membrane properties are largely conserved across the three model membranes. The molecular models and insights emerging from the present work should aid in the development of novel antimicrobials against Gram-positive strains.

Lipid droplets (LDs) are cellular organelles responsible for lipid storage and metabolism. The mechanism of biogenesis of LDs involves phase separation of neutral lipids from the surrounding phospholipids, which generates oil lenses embedded in lipid bilayers, also known as nascent LDs. As nascent LDs grow, at some point they bud out of the bilayer, forming nearly spherical droplets. Nascent LDs have different propensity to bud, and it has been proposed that their shape provides information on such propensity; however, LD shape is difficult to determine experimentally. Here we studied the shape of lipid droplets using MD simulations at the coarse-grained level, and compared it to the predictions by an established theory. Our general system setup features an oil lens embedded into a flat, periodic bilayer. We found that the shape of simulated nascent LDs resembles a spherical cap (i.e., it has constant curvature over most of the surface), in excellent agreement with the theory, already for very small droplet sizes. The aspect ratio (height/radius) of nascent LDs increases with increasing LD volume, increasing membrane softness, and increasing surface tension between oil and water, also in agreement with theoretical predictions; however, it remains lower than 1 (i.e., the ratio for a sphere) for LDs of up to 40 nm in diameter. Fitting the simulated LD shapes with a theoretical shape equation suggests that a non-zero surface tension is present in both the monolayer and in the bilayer region. The existence of a relatively high surface tension in the bilayer region is confirmed by local stress calculations, and indicates that the periodic system setup does not reproduce the properties of nascent LDs in the endoplasmic reticulum, where the bilayer tension is two orders of magnitude lower. However, the simulations provide a microscopic view into the properties of droplet embedded vesicles.

Integral membrane proteins remodel the surrounding lipid bilayer, but quantifying the resulting deformations and linking them to protein density in the membrane has remained challenging. Here, we introduce an integrative methodology that combines all-atom molecular dynamics (MD) simulations with multiscale small-angle X-ray scattering (SAXS) analysis to connect membrane strain to the protein/lipid ratio in proteoliposomes. Using outer membrane phospholipase A (OmpLA) reconstituted into lipid bilayers with both increased and decreased hydrophobic thickness, we systematically probe the effects of positive and negative hydrophobic mismatch.MD simulations demonstrate that OmpLA causes anisotropic, oscillatory thickness deformations extending up to eight times the radius of the first lipid shell surrounding the protein, yet the net change in average membrane thickness remains below 1%. Through our multiscale SAXS analysis, we quantitatively extract structural parameters, ranging from proteoliposome size to internal membrane architecture, using constrained Bayesian inference, with priors derived from MD findings. Specifically, we determine the protein/lipid molar ratio and average membrane strain, revealing excellent agreement between experiment and simulation. In thinner bilayers, substantial protein loss limits the analysis, highlighting the role of bilayer stability in sample preparation. Moreover, the predominance of OmpLA monomers in the thicker membranes is consistent with weak, membrane-mediated repulsive interactions between protein inclusions. Collectively, this integrative approach establishes a framework for quantifying protein-lipid interactions across molecular and mesoscale dimensions.

Plasma membrane sheets generated by controlled mechanical disruption provide direct access to the cytosolic face of the plasma membrane while preserving the native organization of membrane-associated proteins and lipids. Here, we present a protocol for generating and validating sonication-derived plasma membrane sheets from cultured cells, primary neurons, and isolated synaptosomes. We further describe their application for live and fixed imaging of membrane protein localization, organization, conformational dynamics, and protein-protein interactions, as well as quantitative membrane-associated synaptic vesicle recruitment assays. This versatile platform preserves the native membrane environment while enabling direct visualization and quantitative analysis of membrane-associated processes at high spatial resolution. The protocol can be readily adapted to investigate diverse membrane proteins, lipid-dependent mechanisms, and vesicle tethering events across a wide range of cellular systems.

Images in the electron microscope are formed by electron scattering and focusing. The spherical geometry of these processes gives rise to two coherent, conjugate spherical wave fronts, known as Ewald spheres. These spheres are associated with the two halves of the contrast transfer function (CTF), and their widths are determined by the focal gradient through the specimen. To properly correct for the CTF, each half of the CTF must be applied to an image individually and integrated into the reconstruction into the corresponding Ewald sphere. Theory indicates that this dual Ewald sphere reconstruction method should recover the maximal amount of information possible. This method was compared to the other reconstruction methods commonly used: the projection approximation (ignoring the Ewald sphere), the simple insertion and the single sideband methods. In simulated reconstructions the dual Ewald sphere method recovered the most information when the correct half of the CTF is matched to the corresponding Ewald sphere. If the wrong half is matched, the result worse than the projection approximation method. Examining reconstructions from real data indicated that the dual Ewald sphere method performs at least as well as the simple insertion method, but not as good as in simulations. The likely reason is the two-fold ambiguity in the assigned orientations of the particle images, which remains an issue to pursue in further studies. In conclusion, the dual Ewald sphere reconstruction method may offer the best way to calculate very high resolution reconstructions when the micrograph quality warrants it. HighlightsO_LIThe dual Ewald sphere reconstruction corrects for the two halves of the CTF. C_LIO_LIThe signs of the two halves of the CTF must correspond to the focal gradient. C_LIO_LIDetermining the focal gradient for individual particle images remains unresolved. C_LIO_LIComplex reconstructions indicate any real space phases are artifacts. C_LI

Staphyloxanthin (STX) is a carotenoid synthesized by the human pathogen Staphylococcus aureus. The golden color of this bacterium is due to this carotenoid. STX protects Staphylococcus aureus from oxidative stress by scavenging free radical species. Furthermore, STX has been shown to mechanically strengthen the Staphylococcus aureus membrane and to form microdomains that recruit antibiotic-resistance factors. Thus, inhibition of STX is a promising strategy for intervening against multidrug-resistant strains of this pathogen. However, the molecular mechanisms by which STX regulates the membrane structure and function of Staphylococcus aureus remain unclear. More specifically, the localization of STX within phosphatidylglycerol (PG) bilayers, the primary phospholipid of this bacteriums membrane, and how this localization drives macroscopic biophysical changes remain unresolved questions. Here, we addressed this issue by integrating molecular dynamics (MD) simulations, X-ray scattering experiments, and fluorescence spectroscopy. We developed an atomistic model of STX, which was validated against X-ray scattering data and which is suitable for all-atom MD simulations. We demonstrate that STX significantly increases lipid packing and acyl chain order of STX-PG bilayer mixtures. In addition, STX self-assembles into clusters, where the long and rigid conjugated triterpenoid chain interdigitates across both leaflets, modifying locally the density of the surrounding PG molecules. These findings provide a molecular explanation to the reduced headgroup spacing and core dynamics observed in fluorescence experiments and are consistent with the formation of structurally-distinct STX-enriched microdomains. Notably, STX reduces the gel-to-liquid crystalline phase transition temperature, indicating a general stabilizing effect for the fluid phase of PG lipids of varying length. Overall, our findings provide molecular insights into how STX enhances membrane mechanical integrity. It will be highly interesting to establish how the membrane remodeling effects observed here connect with STXs dual roles, acting as an antioxidant and preventing pore formation and other mechanical perturbations induced by antimicrobial molecules.

Molecular dynamics simulations of nucleic acids are performed using a solvent-buffer distance of 10 [A] between the solute surface and the simulation box boundary. Although this cell size has been extensively explored in protein simulations, its implications for nucleic acid dynamics are not well understood. Nucleic acids are elongated, highly charged, and flexible structures with hydration and dynamical properties distinct from those of proteins and therefore, they may require different solvent-layer considerations in simulations. In this study, we investigated the effect of simulation cell size on nucleic acid dynamics by simulating a 30-base-pair double-helical nucleic acid structure and its two single-stranded forms using solvent-buffer distances of 3, 5, 10, 15, and 20 [A]. Smaller cells may impose restricted hydration, molecular crowding, and periodic image interactions. However, larger cells provide solvent space for conformational relaxation. A total of 45 s of molecular dynamics simulations were performed (3 structures x 5 cell sizes x 3 replicates x 1 s). Our results show that while the commonly used 10 [A] buffer may be sufficient to maintain the stability of the double-stranded nucleic acid, larger cells are required to capture the conformational dynamics of single-stranded structures. In both, increasing the cell size to 15 or 20 [A] enables broader conformational sampling. The first hydration shell exhibits reduced crowding in the 20 [A] cell, consistent with more relaxed conformations. At larger cell sizes, single-stranded nucleic acids adopt compact, self-associated conformations for stability. Together, this study presents physical insight into how simulation cell size and solvent environment influence nucleic acid dynamics.

Artificial lipid droplets (aLDs) provide a controllable platform for studying lipid biochemistry, but their use is limited by contamination with other membrane structures and the lack of quantitative methods to assess sample purity. Here, we establish dithionite quenching of NBD-labeled lipids as a simple approach to evaluate aLD purity. The approach relies on dithionite's ability to selectively quench NBD fluorophores exposed in the phospholipid monolayer of aLDs and in the outer leaflet of liposome bilayers, but not those protected within the inner leaflet of liposome bilayers. Consistent with liposome contamination, bulk aLD preparations exhibit incomplete quenching, which can be separated by sucrose gradient centrifugation into liposome-like and droplet-enriched populations based on quenching behavior. Guided by this assay, sonication conditions were optimized to increase aLD purity and reduce liposome contamination. A biotin-streptavidin immobilization strategy further enabled stable imaging of individual aLDs. Finally, we applied this method to probe the accessibility of neutral lipids within aLDs. This revealed hydrophobicity-dependent quenching kinetics of neutral lipids, with less hydrophobic diacylglycerols showing greater surface exposure within aLDs than more hydrophobic triacylglycerols and cholesterol esters. Taken together, these establish dithionite quenching of NBD-labeled lipids as a simple quantitative method for assessing aLD purity and demonstrate its utility for studying lipid accessibility.

Interactions between proteins and glycosphingolipids (GSLs) regulate various cellular processes and altered GSL metabolism contributes to numerous diseases. The diverse glycan headgroups and ceramide backbones of GSLs shape membrane organisation, fluidity, curvature, and tension. As protein recognition frequently depends on both glycan specificity and the organisation of GSLs within the membrane, these interactions remain challenging to characterise in vitro. Here, we introduce FLiPA (Fluorescent Liposome Plate Assay), a versatile method that utilises fluorescent agarose-embedded giant liposomes for the quantitative analysis of protein-GSL interactions. By enabling systematic control of membrane and buffer composition, FLiPA provides an accessible and robust platform for dissecting the molecular determinants of protein-GSL interactions, including the roles of cholesterol, membrane order, protein oligomerisation and ionic strength.

The cryptococcal polysaccharide capsule is a unique eukaryotic virulence factor that is a target for the immune system and the development of therapeutic antibodies. Our understanding of capsular architecture is limited to a few studies suggesting that metal dications play a role. In this work we explore a mechanism of cryptococcal aggregation that depends on calcium phosphate precipitation. We describe the chemical and biophysical properties of calcium interaction with the predominant cryptococcal polysaccharide, glucuronoxylomannan (GXM). We show that cell aggregation is a pH-dependent and occurs in a calcium dose-dependent manner. Furthermore, this cellular aggregation phenomenon as well as interpolymer capsular polysaccharide interactions are unique to calcium dications and do not occur with other mono- or dications as shown by size exclusion chromatography and circular dichroism. Diffusion ordered spectroscopy nuclear magnetic resonance and ab-initio calculations support complexation of calcium with glucuronic acid (GlcA). The ab-initio calculations also suggest that calcium ions can complex up to four GlcA monomers. Not only does calcium act as a scaffold for the cryptococcal capsule, interacting with up to four glucuronic acid residues of GXM, but calcium phosphate treatment of cells reduces the anti-phagocytic properties of the capsule, promoting ingestion by macrophages and altering antibody interactions with the capsule. This work advances our understanding of the cryptococcal capsule, its biophysical properties, by providing a model for the critical role of calcium interactions with capsular polymers of Cryptococcus neoformans including important impacts at the host-cell interface.

Members of the transforming growth factor-{beta} (TGF-{beta}) superfamily, including inhibins and activins, are structurally related glycoprotein dimers that regulate reproductive and endocrine signaling. Their high degree of molecular similarity presents challenges for label-free analytical discrimination. To evaluate the ability of Raman spectroscopy to distinguish closely related TGF-{beta} superfamily proteins based on intrinsic vibrational fingerprints. Raman spectra of recombinant human Inhibin -subunit, Inhibin B ({beta}B homodimer), and Activin A ({beta}A--{beta}A) were acquired using confocal Raman microscopy with 532 nm excitation. Spectra were baseline-corrected, area-normalized, and analysed using principal component analysis (PCA). Distinct spectral signatures were observed across the 500--1800 cm-1 region. Differences within the S--S stretching region (500--550 cm-1) were consistent with variations in disulfide-bond environments, with the Inhibin -subunit exhibiting the highest relative intensity in this region. Variations in the amide I band (1600--1700 cm-1) suggested differences in protein secondary structure, while aromatic amino acid vibrations provided additional discriminatory features. PCA revealed clear clustering and separation of all three protein classes based on their Raman fingerprints. Raman spectroscopy enables label-free differentiation of structurally related endocrine glycoproteins and demonstrates potential for the structural characterization and classification of inhibin and activin proteins within the TGF-{beta} superfamily.

Background: Gadolinium-based contrast agents (GBCAs) are routinely used in magnetic resonance imaging (MRI). Although macrocyclic GBCAs were initially considered biologically inert, it is now known that a fraction of patients retains gadolinium (Gd) for prolonged periods in tissues such as blood, bone, and brain. Because the first cellular interactions of GBCAs occur in the bloodstream, this study aimed to elucidate the uptake mechanism but also the intracellular persistence and release dynamics of gadoterate meglumine, one of the most widely used macrocyclic agents, in white blood cells (WBCs). Methodology and principal findings: WBCs and K562 cells were incubated with gadoterate meglumine under different conditions to investigate its cellular entry mechanisms. Uptake of the contrast agent was quantified by measuring intracellular Gd using single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS). Time and concentration-dependent incubation of K562 cells revealed saturable uptake kinetics consistent with a Michaelis-Menten model which is independent of the phase of the cell cycle. Gadoterate meglumine uptake in both WBCs and K562 cells was shown to be an active process, as uptake was strongly reduced or abolished at low temperature (16C and 4C) and in the presence of metabolic inhibitors (sodium azide and 2-deoxyglucose). Co-incubation with multiple endocytosis inhibitors (Dyngo 4a, Dynole 2-24 and chlorpromazine) did not significantly decrease intracellular Gd levels in K562 cells and caused only a slight reduction in WBCs, indicating that endocytosis is not the main entry pathway for gadoterate meglumine in these cells. Furthermore, we assessed the retention time of the Gd inside the cells, showing that only after 24 hours post incubation 80% percent of the intracellular Gd was released through an active process. Finally, we demonstrate that one of the mechanisms of Gd release from WBCs involves extracellular vesicles, which may substantially increase its potential for downstream accumulation in different tissues, including immunoprivileged tissues like brain. Significance: The observed time-dependent accumulation, temperature and energy dependence of gadoterate meglumine uptake demonstrate that active cellular mechanisms are primarily responsible for GBCA internalization. Furthermore, our results indicate that macropinocytosis, phagocytosis, and clathrin-mediated endocytosis are not the primary routes of gadoterate meglumine entry. Hereby, we also describe that Gd externalization is an active process involving extracellular vesicles which may influence the Gd distribution in different tissues and its consequent long-term retention. Further studies are required to explore strategies to block this process in order to mitigate potential long-term gadolinium retention.

Biological membranes contain a diverse set of membrane proteins surrounded by many different lipids, and the lateral organization and function of these molecules are closely intertwined. Here, we use coarse-grained molecular dynamics (MD) simulations to explore how hydrophobic mismatch between the length of transmembrane (TM) proteins and the thickness of the surrounding lipid membrane impacts the spatial distribution of the lipids. We constructed idealized cylindrically symmetric proteins, inspired by the Mattress Model developed in the 1980s, and simulated these model proteins in different lipid compositions. We found that unsaturated lipids were attracted to short TM proteins that thinned the membrane, while fully saturated lipids were attracted to long TM proteins that induced membrane extension. A simple mechanical description of the membrane deformation energy coupled to a lipid mixing model accurately predicted the enrichment/depletion, which was up to 33% in some cases. Our simulations also highlight that lipid sorting behavior is sensitive to protein tilt and protein surface roughness. By teasing out the fundamental physical principles in these simple models, our results provide a foundational understanding of how proteins and lipids form complex and transient assemblies, which we believe will be important for interpreting lipid-protein interactions for a host of membrane proteins that regulate cellular membranes and cell function.

Weak magnetic fields affect many biological processes across the tree of life, though the precise molecular sensors and pathways involved in such magnetoresponses remain mostly uncharacterized. Fluorescence is a useful tool for investigating magnetic field effects in flavoproteins, as their chromophores fluorescence intensity can be shown to depend on the spin states of electronic radical pairs. Here, we describe a four-state ordinary differential equation model to understand what parameter sets result in fluorescence contrast between spin states in photocycles with singlet and triplet radical pairs. We conclude that only certain sets of parameters result in the fluorescence intensity being a good proxy measurement for singlet yield. In particular, we observe that the illumination intensity required to obtain fluorescence contrast depends on the rate of the slow spin-independent radical termination reactions that recover ground-state oxidized fluorophores. Moreover, to observe a magnetic field effect in fluorescence intensity when an external magnetic field modulates the singlet yield, the illumination intensity must be strong enough such that photoexcitation is not the rate-limiting step. This understanding suggests that flavoproteins that do not exhibit magnetic field effects in their fluorescence emission under certain experimental setups may still be sensitive to weak magnetic fields in terms of function, as magnetosensitivity in fluorescence depends strongly on illumination conditions.

The translocase of the outer mitochondrial membrane (TOM) complex is the main entry gate for mitochondrial proteins. Approximately 99 % of mitochondrial proteins are synthesized as precursor proteins (preproteins) in the cytosol and subsequently translocated into mitochondria through the TOM complex. The TOM complex exists in a dynamic equilibrium among multiple assembly states through spatial rearrangements of its subunits. The recent cryo-electron microscopy (cryo-EM) studies revealed near-atomic structures of the TOM core dimer, whereas previous biochemical studies indicated the TOM complex functions as a trimer in intact mitochondria. However, the relationship between the core dimer and the functional trimer remains unclear. In the present study, we analyzed the dynamics of the TOM complex using high-speed atomic force microscopy (HS-AFM) to investigate the assembly states and conformation transitions of the TOM complexes. We demonstrated that purified yeast TOM complexes predominantly adopt a trimeric organization but dynamically dissociate into dimeric and monomeric states during HS-AFM observation. The trimeric particles observed by HS-AFM exhibited spherical molecular shapes consistent with a trimeric structural model proposed from previous crosslinking analyses. In contrast, the dissociated dimeric particles closely resembled the dimensions of the TOM core-dimer structures determined by cryo-EM. Furthermore, HS-AFM analyses provided insight into the spatial arrangement of the Tom20 receptor, consistent with previous models of the trimeric TOM complex. These observations enabled characterization of the trimeric TOM complex in vitro and provide a foundation for future structural and functional analyses of TOM complex assembly.

Vascular endothelial cells (ECs) form a monolayer lining blood vessels and serve as a barrier between blood and tissues. Clathrin-mediated endocytosis (CME) is a major internalization pathway that involves a physical conformational change of the plasma membrane to form a vesicle and is therefore sensitive to the local environment. ECs are subjected to a myriad of fluid shear stress (FSS) rates from circulating blood, which we hypothesize affects CME. To test this, we used simultaneous two-wavelength axial ratiometry (STAR) microscopy, which provides nanoscale axial resolution, to determine the frequency and morphology of clathrin-coated vesicles as they form. Human umbilical vein endothelial cells (HUVECs) were transfected with dual-tagged clathrin light chain a (CLCa-iRFP-EGFP) and cultured under 10 dyn/cm2 FSS. CME activity was elevated in cells cultured under flow and assayed in static or flow conditions compared to statically cultured and imaged controls, indicating that FSS-induced changes to CME were maintained shortly after flow cessation. Single vesicle analysis showed cells cultured in FSS had a slight preference for vesicle formation with a flat-to-curved clathrin transition compared to control. Next, to assess the impact of different FSS rates, we cultured HUVECs at 20 and 40 dyn/cm2 FSS. We found total CME frequency was elevated compared to control at 20 dyn/cm2, but not 40 dyn/cm2. HUVECs cultured at both 20 and 40 dyn/cm2 had vesicles with increased lifetime and enhanced stability, as well as a higher proportion of vesicles formed through a flat-to-curved transition of clathrin.

Chemical exchange saturation transfer (CEST) has become an indispensable NMR method to characterize slow exchange affecting biomacromolecules, especially for cases involving exchange between a major state and a minor state, the latter of which is often invisible in the spectrum. The CEST method is based on successive irradiation of selective regions of the NMR spectrum using a weak radiofrequency field, B1, while observing the effect on the visible major state when the B1 field saturates the invisible minor state. The need for selective saturation of narrow spectral regions has to date required acquisition of many tens of two-dimensional CEST spectra to sample the entire spectrum with sufficient resolution. Here we present the ACCEST method which measures an entire CEST profile from a single two-dimensional accordion-CEST spectrum plus a reference spectrum. ACCEST is based on the concept of accordion spectroscopy, where in the present implementation the carrier frequency of the weak saturating B1 field is stepped in synchrony with the dwell-time incrementation in the indirect dimension of the two-dimensional spectrum. We benchmarked ACCEST against conventional CEST, resulting in excellent agreement for both backbone 15N and methyl 13C CEST profiles. ACCEST offers substantial time savings that scale linearly with the number of spectra required in the corresponding conventional CEST experiment. Thus, ACCEST can dramatically speed up lengthy serial experiments, such as ligand titrations or temperature-dependent studies, and enable studies of non-equilibrium systems or samples with limited lifetimes.

Human aquaporins (AQPs) are essential membrane channels, yet their inherent hydrophobicity complicates structural and functional studies. We present the systematic application of the QTY code to human AQPs, integrating it with AlphaFold 3 structure prediction to design and validate that four-representative human AQPs (AQP1, AQP3, AQP4, AQP7) can be converted into water-soluble analogs while maintaining their conformation. This approach features a novel platform for editing challenging membrane proteins. The QTY code was applied to the transmembrane regions of the selected four AQPs. Subsequently, the water-soluble QTY analogs of the four AQPs were predicted using AlphaFold 3. The predicted structures were superposed with CyroEM- or X-ray-determined native structures in PyMOL. Further analyses included root-mean-square deviation (RMSD) calculations, visualization of hydrophobic surface reduction, and inspection of conserved protein-ligand binding ability. After applying the QTY code, sequence changes between native AQPs and their QTY analogs was significant (42.86-48.80%). Nevertheless, their structures superposed well in analyses, with only slight deviations (RMSD < 0.6 [A]). In addition, the surface hydrophobicity of all QTY-edited AQPs was significantly reduced. Importantly, molecular contacts between the cholesterol ligand and protein were largely preserved for both native AQP1 and its QTY analog. Finally, all AlphaFold3-predicted structures for AQPs have high confidence values (pLDDT > 90; pTM ~0.83), supporting the reliability of the predicted structures. The findings demonstrate that membrane protein hydrophobicity can be edited and reduced without compromising fold integrity or functional architecture. Integration of the QTY code with AlphaFold 3 affords a high-throughput platform for designing water-soluble, structurally faithful analogs of challenging membrane proteins. Such a strategy can provide a potent platform for detergent-free biochemical studies and water-soluble analogs for therapeutic monoclonal antibody discoveries, thus advancing research of this pharmacologically important protein family.

Poly-L-Lysine (PLL) mediates the non-specific adhesion of cells and is commonly used in Atomic Force Microscopy (AFM) measurements, to ensure that cells remain attached to the substrate. However, it is acknowledged that adhesion affects the measured mechanical properties, in particular in the case Red Blood Cells (RBCs). This results in a wide range of Youngs modulus E reported in the literature. The present study aims at providing a systematic approach to the impact of non-specific adhesion on the rheology of RBCs. It provides a correlation between the topography profile of adherent RBCs and their rheology, from weak (cPLL = 10-3 mg/mL) to strong-adhesion (cPLL = 100 mg/mL) regimes. Using RICM and AFM, we find that there is a continuum of RBC shapes promoted by adhesion, from concave to dome-shaped, as predicted by the theory of vesicle adhesion. Their elastic properties discriminate them into two populations depending on adhesion strength, where stiffer RBCs (E {gtrsim} 100 Pa) correlate with dome-shaped cells. These findings are supported by rheology measurements of the dynamic complex shear modulus G*(f): while the storage modulus increases with cell-substrate adhesion, reflective of an increased membrane shear modulus, the loss modulus remains unchanged. Finally, further analysis inspired by membrane theory shows that different deformation modes may be triggered during indentation of either weakly or strongly adhering RBCs, illustrating the limits of the Hertz model.