The central amphipathic region of perilipin 1 (PLIN1) contributes to lipid-droplet targeting, but the interfacial properties that govern its association with lipid assemblies remain incompletely defined. Here, we examined human PLIN1 residues 94-193 (PLIN1-AH) by solution nuclear magnetic resonance spectroscopy in dodecylphosphocholine (DPC) micelles and dimyristoyl phosphatidylcholine/dihexanoylphosphatidylcholine (DMPC/DHPC) bicelle-forming assemblies, complemented by biolayer interferometry (BLI) measurements of DMPC/dimyristoyl phosphatidylglycerol (DMPG) large unilamellar vesicle recruitment to immobilized PLIN1-AH. DPC micelles produced extensive spectral reorganization, whereas neutral DMPC/DHPC bicelles caused comparatively modest perturbations. At q = 0.8, increasing DMPG from 25 to 50 mol% shifted the NMR response from a low- to a high-response regime, with no further increase at 75-100 mol%. At 50 mol% DMPG, increasing q to 1.6 further enhanced the spectroscopic response. Vesicle recruitment showed a corresponding nonlinear composition dependence, reaching its maximum at 50 mol% DMPG among the compositions tested. These results indicate that PLIN1-AH association is sensitive to both membrane mimetic organization and anionic-phospholipid content and support a threshold-like transition around 50 mol% DMPG under the conditions tested.

The transbilayer distribution of cholesterol in the plasma membrane is unresolved in that diverse analyses have yielded contradictory results. We now propose a model based on a novel mechanism. It assumes that (a) sterols associate stoichiometrically with phospholipids and (b) these complexes are filled to capacity in the plasma membrane. The sterol in each leaflet is then the product of the abundance of the leaflet phospholipids and their sterol stoichiometries. We applied the model to the bilayer of the human erythrocyte utilizing literature values for its cholesterol content, the abundance of phospholipids in the two leaflets and their cholesterol: phospholipid stoichiometries (1:1 mole/mole in the exofacial leaflet and 1:2 mole/mole in the endofacial leaflet). The model predicts that the outer leaflet contains about two thirds of the cholesterol in this membrane; i.e., twice that in the inner leaflet. Molecular dynamics simulations have delivered similar sidedness values for membranes in which the exofacial phospholipids have a higher sterol affinity. However, our mechanism holds that sterol affinity is irrelevant if the phospholipids are replete with sterol. Our results meet the requirement that the areas of the two leaflets be about equal. The model predicts that the sterol abundance in one leaflet of a bilayer will exceed that in the other when its phospholipids are fully complexed and have a higher sterol stoichiometry.

Muscle relaxation is enabled by the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), which removes calcium from the cytosol and returns it to the lumen of the sarcoplasmic reticulum. During transport, SERCA adopts a variety of conformational states which differ in their structure and affinity for substrates. The transition between these states is mediated by calcium and ATP binding and the formation of an aspartyl-phosphate intermediate, which enables the transport of calcium across the membrane. SERCA function is highly regulated because of the importance of calcium in processes such as muscle contraction-relaxation. A family of tissue-specific transmembrane regulatory subunits interact with SERCA, exemplified by sarcolipin (SLN) in skeletal muscle and phospholamban (PLN) in cardiac muscle. SLN and PLN are known to alter the apparent calcium affinity and maximal activity of SERCA. In the present study, we investigated SLN inhibition of SERCA under conditions that varied the substrate-dependent conformational state of SERCA. Measuring both calcium-dependent ATP hydrolysis and charge translocation, we found that SLN inhibition was dependent on the initial state of SERCA. Under substrate conditions that poised SERCA in the calcium-free E2 state, SLN was more inhibitory and impacted both the maximal activity and apparent calcium affinity of SERCA. In contrast, SLN inhibition was reduced under pre-incubation conditions that favored the calcium-bound E1 state of SERCA. We conclude that SLN is capable of distinct modes of interaction with SERCA depending on the conformational state, and that the mode of interaction exhibits conformational memory in that the initial state persists during steady-state turnover of SERCA.

We developed a nanodisc (ND)-based platform with recombinant acid sphingomyelinase (ASM) embedded in a bilayer of phospholipids that are circumscribed by apolipoprotein E3 (apoE3) for targeted delivery to lysosomes. We designed a fusion protein comprising essential catalytic segments of ASM with a transmembrane helix at one end (TM-ASMcat). Purified TM-ASMcat was reconstituted with phospholipids and the N-terminal (NT) domain of apoE3 to promote the formation of NDs (TM-ASMcat-ND). Physicochemical characterization revealed the presence of both TM-ASMcat (35 kDa) and apoE3NT (24 kDa) in the preparations, and formation of large complexes (400-600 kDa). Transmission electron microscopy of TM-ASMcat-ND revealed discoidal complexes (major/minor axes of 30.9 ± 11.9/8.6 ± 2.0 nm, respectively), while atomic force microscopy showed NDs ~ 14 nm high. The TM-ASMcat-ND displayed a significant ability to hydrolyze sphingomyelin at pH 5. LC-MS/MS of glioblastoma cells treated with TM-ASMcat-ND revealed a significant decrease (22.3%) in total sphingomyelin content compared to cells treated with PBS. Sphingolipidomic analysis showed a trend of decrease in specific sphingomyelin species compared to empty ND-treated cells. Immunofluorescence analysis revealed perinuclear vesicles showing colocalization of ASM with apoE3 and Lysotracker, indicative of successful targeting of TM-ASMcat-ND to the lysosomes. Taken together, our data indicate that TM-ASMcat has been embedded in ND, and that it retains catalytic activity in solution and inside the cells. Our findings bear the potential to treat ASM deficiency in Niemann-Pick disease Type A and B, which are lysosomal storage disorders characterized by the accumulation of sphingomyelin.

The global challenge of increasing antibiotic resistance development has necessitated the continued development and investigation of novel and existing antimicrobials. Daptomycin is a cyclic lipopeptide isolated from Streptomyces roseosporus, which has been clinically approved to treat a variety of gram-positive infections. Daptomycin is a membrane-active antimicrobial peptide that requires Ca[2+] ions and phosphatidylglycerol lipids to exert its antimicrobial activity. To better understand the mechanism of action at the biophysical level, daptomycin interactions with bilayers and bacterial membranes were evaluated across different solution pH values and by varying cholesterol content in the model membranes. The results demonstrate that pH 5-8 does not affect daptomycin binding to model membranes or its structural rearrangement upon binding to the bilayer. Similarly, including cholesterol up to 30 mol% in the bilayers did not affect daptomycin binding, with an apparent binding affinity of ~ 1 µM under these conditions. Fluorescence quenching experiments showed that Ca[2+] binding to daptomycin induced a small but significant shift in the position of Trp in the bilayer, becoming more deeply inserted when calcium was present. Bacterial membrane permeabilization assays confirmed that daptomycin forms stable pores in bacterial membranes large enough to cause leakage of the chromogenic substrate, o-nitrophenyl-β-D-galactopyranoside (ONPG). The data demonstrate a remarkable robustness of daptomycin interactions with lipid bilayers.

Life requires membranes. But what were the first membranes made of, and how did they acquire the functional complexity of modern cells? Here we show that nonanoic acid and nonanol, two short-chain amphiphiles found in carbonaceous meteorites, spontaneously incorporate gangliosides into their membranes, generating a ternary vesicle system that, under prebiotic-inspired conditions, recapitulates three hallmarks of eukaryotic membrane biology: (i) lateral membrane domain segregation consistent with lateral compositional heterogeneity analogous to liquid-ordered and liquid-disordered phase co-existence; (ii) ganglioside-dependent and charge-selective capture of cationic peptides, confirmed by both centrifugation-binding assays and Langmuir monolayer experiments; and (iii) inward membrane budding and intraluminal vesicle formation upon cationic peptide treatment, recapitulating multivesicular body biogenesis in a minimal molecular system. Molecular modeling reveals that the nonanoic acid/nonanol pair occupies a molecular volume equivalent to the sterane core of cholesterol, explaining its cholesterol-surrogate function. These findings demonstrate that lateral domain segregation, surface glycolipid asymmetry, and endocytic membrane remodeling are not evolutionary inventions; they are emergent physicochemical properties of amphiphile-glycolipid systems that predate cellular life itself.

P4‑ATPase lipid flippases establish transbilayer lipid asymmetry in eukaryotic membranes, with increasing evidence indicating a role for interacting partners in regulating flippase activity. However, assessing interactor‑dependent effects on flippase activity in mammalian cells is complicated by heterogeneous protein expression and background lipid transport. Here, we introduce an expression‑based gating strategy for in‑cell NBD‑lipid uptake assays that improves sensitivity and interpretability under transient expression conditions. Using this approach, we examined the influence of the putative interactors VAMP3 and TMEM230 on the ability of ATP11C to transport phosphatidylserine (PS). VAMP3 exhibited no detectable effect on NBD‑PS uptake, whereas TMEM230 produced a strong inhibitory phenotype. The expression‑based strategy increased resolution between ATP11C variants and substantially reduced the number of events required for reliable analysis. Together, these advances provide a framework for systematic, cell‑based investigation of regulatory interactions governing mammalian P4‑ATPase function.

Lipid oxidation plays an important role in modifying membrane organization and interfacial behavior, particularly in phospholipids containing unsaturated acyl chains. In this study, Langmuir monolayer measurements and thiobarbituric acid reactive substances (TBARS) analysis were used to investigate the oxidative behavior of saturated and unsaturated phosphatidylcholine model systems. Surface pressure-mean molecular area (π-Mma) isotherms and constant-pressure stability measurements were obtained for DMPC, DPPC, POPC, and PAPC monolayers under ambient and controlled oxidative conditions. Differences in acyl-chain saturation influenced phospholipid packing behavior, monolayer organization, and oxidative response under interfacial conditions. Controlled oxidation using the radical initiator 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) induced distinct structural responses in PAPC monolayers relative to measurements performed under ambient conditions. TBARS analysis confirmed greater oxidative susceptibility in unsaturated phospholipids, with PAPC exhibiting substantially higher malondialdehyde formation than POPC. Addition of α-tocopherol reduced TBARS formation in oxidized PAPC vesicles, consistent with its established antioxidant role in polyunsaturated lipid systems. These findings demonstrate how phospholipid unsaturation influences monolayer organization and oxidative response under interfacial conditions while highlighting the complementary use of Langmuir and TBARS approaches for investigating oxidation-dependent membrane behavior.

Replacing a protein's sidechain can alter its interaction with a membrane, conferring a thermodynamic benefit or penalty. These thermodynamic consequences of mutations can be estimated using hydrophobicity scales, yet are challenging to validate experimentally. Here, we examine the structural and thermodynamic consequences of membrane interactions using two model peptides KALP23 and WALP23. In these peptides, respectively, two lysine residues or two tryptophan residues are placed at each end of a transmembrane helix. Application of several hydrophobicity scales suggests that the free energy of insertion of KALP23 is 10-15 kcal/mol less favorable than that of WALP23. According to these predictions, the lowest free energy for KALP would be in solution and not in the membrane, and KALP would be kinetically "trapped" in the transmembrane conformation during standard sample preparation based on co-solubilization with lipids in organic solvents. In order to quantitatively test this prediction, we utilized a different approach of sample preparation which utilizes fluorinated surfactants capable of maintain hydrophobic peptides in solution without interacting with the bilayer themselves. We demonstrated that both peptides can be inserted into the preformed vesicles, and that the free energy difference between their partitioning is < 1 kcal/mol. These results disprove the hypothesis that KALP peptides are kinetically trapped and open the doors for using this system for thermodynamic measurements to further test the additivity assumptions implicit in applications of hydrophobicity scales.

The cell surface localization of the Na,K-ATPase (sodium pump) is required for maintaining transmembrane electrochemical gradients. While glycosylation of the β1 subunit facilitates trafficking from the endoplasmic reticulum to the plasma membrane, its role in nanoscale surface organization is not characterized. This study employed GlycoSHIELD computational modeling and DNA-PAINT single-molecule localization microscopy (SMLM) to evaluate how N-glycans influence pump distribution. In-silico simulations indicated that N-glycans sequester the protein core, providing a steric shield that increases with structural complexity. To investigate this experimentally, glycosylation-deficient mutants (3NQ) were generated and confirmed via immunoblotting. Quantitative SMLM analysis of A498 cells demonstrated that wild-type pumps exhibit higher localization density and form larger (144 nm) and more frequent clusters than 3NQ mutants (109 nm). These results indicate that N-glycosylation promotes stable enzyme clustering, supporting a galectin-lattice mechanism of organization rather than steric repulsion.

Time-resolved X-ray solution scattering (TR-XSS) provides direct access to protein structural dynamics but has largely been restricted from the microsecond range up to approximately 100 milliseconds. As a result, slower enzymatic systems, including many P-type ATPases, remain difficult to probe. Here, we extend the temporal reach of TR-XSS by sequentially positioning radiation damage-free acquisition windows to enable capturing structural evolution across sub-second to second timescales. Implemented at the CoSAXS beamline at MAX IV Laboratory, this strategy enables continuous tracking of slow protein dynamics while preserving structural sensitivity. Using adenylate kinase (AdK) as a benchmark, we observed a single conformational transition accompanied by signal amplitude decay. In contrast, application to the prokaryotic P-type ATPase LMCA1 revealed clear evolution in scattering profiles, consistent with sequential conformational transitions. Kinetic analysis identified two transitions on the 140 ms and 660 ms timescales, which correspond monitoring rise and decay of a rate-limiting step which can symbolize intermediate dynamics in a slow transport cycle. The results demonstrate that extended-time TR-XSS can resolve multi-step reaction pathways in slow membrane proteins. The approach broadens the accessible timescale of TR-XSS and establishes a general framework for studying slow conformational dynamics in P-type ATPases and related systems.

The viral envelope fuses with the host cell membrane to initiate viral infection. In the case of SARS-CoVs, the fusion process is catalyzed by the spike (S) protein. The S1 subunit interacts with the host cell receptor and assists in docking the viral particle at the cell surface, whereas the fusion process is induced by the insertion of a conserved hydrophobic motif at the N-terminal of the S2 subunit, known as a fusion peptide, into the host cell. Current experimental findings suggest that the fusion peptide of SARS-CoVs displays a strong Ca[2+]-dependent membrane binding and fusogenic activity, indicating that Ca[2+] functions as a critical cofactor during viral entry. Nevertheless, many earlier studies fail to clearly differentiate between fusion driven directly by Ca[2+] and that mediated by the fusion peptide, often due to the absence of appropriate controls for different Ca[2+] concentrations. In this work, we have assessed the impact of the consensus fusion peptide of SARS-CoVs in polyethylene glycol (PEG)-mediated fusion of small unilamellar vesicles with varying Ca[2+] concentration, where appropriate control experiments have been carried out in the absence of the peptide. Our results show that the peptide has no significant effect on Ca[2+] in inducing lipid mixing of model membranes.

P4-ATPases are lipid flippases that maintain membrane phospholipid asymmetry by transporting specific phospholipids from the exoplasmic to the cytosolic leaflet, an essential process for membrane integrity, trafficking and signaling. Several P4-ATPases are tightly regulated by autoinhibitory N- and C-terminal extensions, yet the molecular basis of this regulation remains incompletely understood. Here, we investigated the autoinhibition mechanism of the human flippase ATP8B1 using trans-inhibition assays with synthetic peptides derived from its C-terminal tail. Using purified C-terminally truncated ATP8B1-CDC50A, we systematically assessed the inhibitory properties of peptides corresponding to distinct segments of the C-terminus. We show that the distal disordered region of the C-terminal tail significantly contributes to autoinhibition, likely through transient interactions with the cytosolic domains. We further identify a critical interaction between R1228 in the C-terminal tail and E219 in the A-domain, whose disruption markedly reduces inhibitory potency. In addition, we demonstrate that a minimal peptide spanning residues 1216-1228, which bridges the A- and N-domains in the autoinhibited conformation, is sufficient to inhibit ATPase activity. Together, these results refine the molecular description of ATP8B1 autoinhibition, open the way for structure-based activation strategies and provide insight into conserved regulatory mechanisms among P4-ATPases.

Cardiotonic steroids (CTS) have gained prominence due to various mechanisms of action proposed or demonstrated to explain their effects, including their known classical and non-classical signaling pathways mediated by Na+/K+-ATPase (NKA) binding. For the characterization, interaction kinetics have been used to better translate in vivo efficacy and selectivity. In addition, temperature effects assessed by thermodynamic analysis provide deeper insight into intrinsic efficacy. However, the impact of conformational changes on both kinetic and thermodynamic parameters of CTS binding has been largely overlooked. This review discusses how these kinetic effects relate to efficacy and selectivity in the experimental characterization of CTS-NKA interactions. We focus on the most straightforward and high-throughput methods for describing CTS-NKA interaction, such as enzymatic activity inhibition, looking at dephosphorylation activity of NKA cycle, and compare them with gold-standard methods and its limitations. Finally, we include some statistical considerations for data analysis and future perspectives. This will provide better insight into the efficacy of this class of inhibitors/agonists targeting the NKA in drug discovery.