Biochimica et Biophysica Acta (BBA) - Biomembranes

SNARE-mediated membrane fusion is regulated by the lipid composition of the engaged bilayers. Lipids impact fusion through direct protein-lipid interactions or through modulating the physical properties of membranes to affect protein function. Lysophospholipids (LPLs) can affect membrane curvature, fluidity and energy of deformation. Their effects are due to their head group, and the length and saturation of their single acyl chains. Here we examined how the properties of LPLs affect yeast vacuole fusion and ion transport. We found that lysophosphatidylcholine (LPC) with acyl chains containing 14-18 carbons inhibited fusion with IC50 values of {cong} 40-120 {micro}M. While acyl chain length moderately affected fusion, the head group played a major role. Unlike LPCs, Lysophosphatidic acid (LPA 18:1) failed to fully inhibit fusion, while lysophosphatidylethanolamine (LPE 18:1) had no effect. Separately we found that changes in acyl chain length and saturation differentially affected Ca2+ transport and vacuole acidification. Together these data show that the effects of LPLs on membrane fusion and ion transport were due to a combination of head group type and acyl chain length.

The structural and dynamic properties of membranes are known to vary with bilayer size and hydration. While Molecular Dynamic (MD) simulations are a powerful tool for studying cellular membrane systems, the results can be sensitive to the analysis work-flow and software. In this study, all-atom MD simulations (500 ns) were conducted on systems of 256, 512, and 1024 POPC lipids at 40, 80, and 160 waters per lipid. With these simulations, a two-fold study was performed: (1) to assess the convergence of structural and dynamic properties of POPC bilayers as a function of membrane size and hydration level using CPPTRAJ (CPP), including area per lipid (APL), bilayer thickness, order parameter, headgroup orientation, and lateral diffusion, and (2) to compare the analysis output and performance of four software packages: CPP, GROMACS (GRO), MDAnalysis (MDA), and LiPyphilic (LiP). For the first objective, our results show that the average values of the bilayer thickness, order parameter, and headgroup orientation are largely independent of the size and hydration levels studied. In contrast, lateral diffusion coefficient was sensitive to both size and hydration. We found that increasing the system size primarily decreased the statistical variance of the APL and thickness. For the second objective, all four packages produced consistent results for APL and thickness, with the most significant discrepancy being a known artifact from the gmx order tool when applied to unsaturated carbons. Performance bench-marks identified CPP as the fastest serial tool for all properties, whereas parallelization benefited MDA and LiP in some metrics. These findings provide a practical roadmap, demonstrating that moderately sized systems (e.g., 256L), combined with an optimized tool such as CPP, offer an efficient workflow for membrane structural property analysis.

We compared the conformations of the transmembrane domain (TMD) of influenza A M2 (IAM2) protein reconstituted at pH 7.4 in DOPC/DOPS bilayers to those in isolated E. coli membranes, having preserved its native proteins and lipids. IAM2 is a single-pass transmembrane protein known to assemble into homo-tetrameric proton channel. To represent this channel, we made a construct containing the IAM2s TMD region flanked by the juxtamembrane residues. The single cysteine substitute, L43C, of leucine located in the bilayer polar region was paramagnetically tagged with a methanethiosulfonate nitroxide label for the ESR (electron spin resonance) study. We compared the conformations of the spin-labeled IAM2 residing in DOPC/DOPS and native E. coli membranes using continuous-wave (CW) ESR and double electron-electron resonance (DEER) spectroscopy. The total protein-to-lipid molar ratio spanned the range from 1:230 to 1:10,400. The CW ESR spectra corresponded to a nearly rigid limit spin label dynamics in both environments. In all cases, the DEER data were reconstructed into the distance distributions showing well-resolved peaks at 1.68 nm and 2.37 nm. The peak distance ratio was 1.41{+/-}0.2 and the amplitude ratio was 2:1. This is what one expects from four nitroxide spin-labels located at the corners of a square, indicative of an axially symmetric tetramer. Distance modeling of DEER data with molecular modeling software applied to the NMR molecular structures (PDB: 2L0J) confirmed the symmetry and closed state of the C-terminal exit pore of the IAM2 tetramer in agreement with the NMR model. Thus, we can conclude that IAM2 TMD has similar conformations in model and native E. coli membranes of comparable thickness and fluidity, notwithstanding the complexity of the E. coli membranes caused by their lipid diversity and the abundance of integral and peripheral membrane proteins.

Model asymmetric bilayers are useful for studying the coupling between lateral and transverse lipid organization. Here, we used calcium-induced hemifusion to create asymmetric giant unilamellar vesicles (aGUVs) for exploring the phase behavior of 16:0-PC/16:1-PC/Cholesterol, a simplified model for the mammalian plasma membrane. Symmetric GUVs (sGUVs) were first prepared using a composition that produced coexisting liquid-disordered and liquid-ordered phases visible by confocal fluorescence microscopy. The sGUVs were then hemifused to a supported lipid bilayer (SLB) composed of uniformly mixed 16:1-PC/Cholesterol. The extent of outer leaflet exchange was quantified in aGUVs in two ways: (1) from the reduction in fluorescence intensity of a lipid probe initially in the sGUV ("probe exit"); or (2) from the gain in intensity of a probe initially in the SLB ("probe entry"). These measurements revealed a large variability in the extent of outer leaflet exchange in aGUVs within a given preparation, and two populations with respect to their phase behavior: a subset of vesicles that remained phase separated, and a second subset that appeared uniformly mixed. Moreover, a correlation between phase behavior and extent of asymmetry was observed, with more strongly asymmetric vesicles having a greater probability of being uniformly mixed. We also observed substantial overlap between these populations, an indication that the uncertainty in measured exchange fraction is high. We developed models to determine the position of the phase boundary (i.e., the fraction of outer leaflet exchange above which domain formation is suppressed) and found that the phase boundaries determined separately from probe-entry and probe-exit data are in good agreement. Our models also provide improved estimates of the compositional uncertainty of individual aGUVs. We discuss several potential sources of uncertainty in the determination of lipid exchange from fluorescence measurements. Statement of SignificanceWe used calcium-induced hemifusion to create an asymmetric lipid distribution in giant unilamellar vesicles that are models for the mammalian plasma membrane. Confocal fluorescence micrographs of asymmetric vesicles showed that coexisting liquid-ordered and liquid-disordered domains initially present in symmetric vesicles were disrupted after 75% of the saturated lipid in their outer leaflets was replaced with unsaturated lipid. We developed quantitative models for extracting valuable information from the data, including the location of the phase boundary and the compositional uncertainty of individual asymmetric vesicles. The methodology we describe can help reveal the molecular determinants of interleaflet coupling of phase behavior and thus contribute to a better understanding of lipid raft phenomena.

The structural and functional characteristics of membrane proteins can be influenced by the composition of the membrane. Consequently, native membranes are most relevant for the study of receptors and other membrane proteins. In this study, we investigated two types of cell-derived vesicles: natively shed extracellular vesicles (EVs) and mechanically derived vesicles (MVs). To this end, we utilized the human breast cancer cell line SKBR3, which strongly overexpresses the receptor HER2. We designed a protocol based on designed ankyrin repeat proteins (DARPins) to purify EVs and MVs enriched in HER2, and to ensure the native orientation of the HER2 receptors within the vesicle. The isolated HER2-containing EVs and MVs were characterized by cryo-EM, cryo-electron tomography (cryo-ET) and mass spectrometry (MS), which revealed fundamental differences between the different vesicle types. Our study highlights the greater structural diversity of EVs over MVs. A single particle cryo-EM analysis and classification of all visible receptors on the vesicle surface yielded electron density consistent with HER2 at modest resolution. Taken together, our results suggest that MVs can serve better than EVs as a suitable platform for the structure determination of membrane proteins within their native membrane environments.

Bicelles have been demonstrated to be a valuable tool for studying membrane protein interactions and structure in vitro. They are distinguished by a distinct lipid bilayer that mimics the plasma membrane of cells making it more native-like than its detergent micelle counter-part. Bicelles are typically comprised of a long-chain phospholipid such as dimyristoylphosphatidylcholine (DMPC) and a short-chain phospholipid such as dihexanoylphosphatidylcholine (DHPC). When mixed together in solution DMPC-DHPC bicelles assume a discoidal structure comprised of a heterogeneous arrangement where the short-chain lipids gather around the rim of the disk and the long-chain lipids form the flat, planar, bilayer region. In this study, the nonionic surfactant, C8E5, was used to prepare mixtures with DMPC to determine if it adopts properties similar to bicelles with a q [≥] 0.5. At q [≥] 0.5, DMPC-DHPC bicelles are bilayered and DMPC is sequestered from the detergent micelle-like DHPC. Mixtures of DMPC and C8E5 were prepared at various q values, a parameter used to describe the mole ratio of DMPC to DHPC in the preparation of bicelles. Employing biophysical methods like dynamic light scattering, 31P-NMR and analytical ultracentrifugation, properties of these lipid-detergent complexes are described. Interestingly they adopted a spherical-shaped micellar structure morphology and did not assume a discoidal shape typical of bicelles at q [≥] 0.5. However, they appear to retain bilayer-like properties that may prove beneficial for in vitro biophysical studies of membrane proteins.

Liposomes are widely used as model lipid membrane platforms in many fields, ranging from basic biophysical studies to drug delivery and biotechnology applications. Various methods exist to prepare liposomes, but common procedures include thin-film hydration followed by extrusion, freeze-thaw, and/or sonication. These procedures have the potential to produce liposomes at specific concentrations and membrane compositions, and researchers often assume that the concentration and composition of their liposomes are similar to, if not identical, to what would be expected if no lipid loss occurred during preparation. However, lipid loss and concomitant biasing of lipid composition can in principle occur at any preparation step due to nonideal mixing, lipid-surface interactions, etc. Here, we report a straightforward method using HPLC-ELSD to quantify the lipid concentration and membrane composition of liposomes, and apply that method to study the preparation of simple POPC/cholesterol liposomes. We examine many common steps in liposome formation, including vortexing during re-suspension, hydration of the lipid film, extrusion, freeze-thaw, sonication, and the percentage of cholesterol in the starting mixture. We found that the resuspension step can play an outsized role in determining the overall lipid loss (up to [~]50% under seemingly rigorous procedures). The extrusion step yielded smaller lipid losses ([~]10-20%). Freeze-thaw and sonication could both be employed to improve lipid yields. Hydration times up to 60 minutes and increasing cholesterol concentrations up to 50 mole% had little influence on lipid recovery. Fortunately, even conditions with large lipid loss did not substantially influence the target membrane composition more than [~]5% under the conditions we tested. From our results, we identify best practices for producing maximum levels of lipid recovery and minimal changes to lipid composition during liposome preparation protocols. We expect our results can be leveraged for improved preparation of model membranes by researchers in many fields. Statement of SignificanceLiposomes are spherical lipid membranes that can be prepared by a variety of biophysical techniques. Researchers use liposomes in a variety of ways, including fundamental biophysical studies of lipid membranes, in drug delivery, drug formulation, and other biotechnology applications. In this report, we study the process to prepare liposomes by several common techniques and validate how reliable each technique is at producing consistent liposome concentrations and lipid compositions. We identify best practices for researchers to produce reliable liposome preparations.

Lipid membranes play an essential role in biology, acting as host matrices for biomolecules like proteins and facilitating their functions. Their structures, and structural responses to physiologically relevant interactions, i.e. with membrane proteins, provide key information for understanding biophysical mechanisms. Hence, there is a crucial need of methods to understand the effects of membrane host molecules on the lipid bilayer structure. Here, we present a purely experimental method for obtaining the absolute scattering length density (SLD) profile and the area per lipid of liposomal bilayers, by aiding the analysis of small angle X-ray scattering (SAXS) data with the volume of bare headgroups obtained from fast (20-120s) grazing incidence off-specular scattering (GIXOS) data from monolayers of the same model membrane lipid composition. The GIXOS data experimentally demonstrate that the variation of the bare headgroup volume upon lipid packing density change is small enough to allow its usage as a reference value without knowing the lipid packing stage in a bilayer. This approach also bares the advantage that the reference volume is obtained at the same aqueous environment as used for the model membrane bilayers. We demonstrate the validity of this method using several typical membrane compositions, as well as one example of a phospholipid membrane with an incorporated transmembrane peptide. This methodology allows to obtain absolute scale values rather than relative scale by using solely X-ray-based instrumentation, retaining a similar resolution of SAXS experiments. The presented method has high potential to understand structural effects of membrane proteins on the biomembrane structure.

Many cytosolic proteins critical to membrane trafficking and function contain an unstructured domain that can bind to specific membranes, with a transition into an amphipathic helix induced upon membrane association. These inducible amphipathic helices often play a critical role in organelle recognition and subsequent function by these cytosolic proteins, but the tools and techniques used to characterize affinity towards specific membranes are low-throughput and highly dependent on the solubility of the inducible amphipathic helix. Here, we introduce a modular recombinant protein platform for rapidly measuring the binding affinity of inducible amphipathic helices towards a variety of membrane compositions and curvatures using high-throughput fluorescence anisotropy measurements. Inducible amphipathic helices are solubilized with a fluorescently tagged small ubiquitin-like modifier (SUMO) protein and binding to membranes quantified by leveraging the unexpected decrease in fluorescence anisotropy upon binding, a phenomenon previously observed but not well understood. By using fluorescence anisotropy decay measurements and solution NMR experiments, we deduce that this phenomenon likely occurs due to the local increase in fluorophore motion upon binding to the membrane. Altogether, this recombinant protein platform can be readily applied to any inducible amphipathic helix of interest, allowing for detailed investigation of the specific membrane biochemical parameters facilitating binding.

The Proton-Coupled Folate Transporter (PCFT) is a transmembrane transport protein that controls the absorption of dietary folates in the small intestine. PCFT also mediates uptake of chemotherapeutically used antifolates into tumor cells. PCFT has been identified within lipid rafts observed in phospholipid bilayers of plasma membranes, a micro environment that is altered in tumor cells. The present study aimed at investigating the impact of different lipids within Lipid-protein nanodiscs (LPNs), discoidal lipid structures stabilized by membrane scaffold proteins, to yield soluble PCFT expression in an E. coli lysate-based cell-free transcription/translation system. In the absence of detergents or lipids, we observed PCFT quantitatively as precipitate in this system. We then explored the ability of LPNs to support solubilized PCFT expression when present during in-vitro translation. LPNs consisted of either dimyristoyl phosphatidylcholine (DMPC), palmitoyl-oleoyl phosphatidylcholine (POPC), or dimyristoyl phosphatidylglycerol (DMPG). While POPC did not lead to soluble PCFT expression, both DMPG and DMPC supported PCFT translation directly into LPNs, the latter in a concentration dependent manner. The results obtained through this study provide insights into the lipid preferences of PCFT. Membrane-embedded or solubilized PCFT will enable further studies with diverse biophysical approaches to enhance the understanding of the structure and molecular mechanism of folate transport through PCFT. HighlightsO_LICell free expression of PCFT without any lipids or detergents resulted in quantitative precipitation of in-situ synthesized PCFT. C_LIO_LIAdditives for expression of PCFT in the soluble fraction were identified. C_LI

Small-angle scattering can be used to derive structural information about membrane proteins reconstituted in suitable carrier systems enabling solubilization of the membrane proteins in question. Since the studies are done in solution, there is no need for crystallization or deposition on sample grids, and it is in principle possible to obtain structural information about intrinsically disordered regions which cannot be resolved by crystallography or the quantitative link to which is hard to establish using e.g. electron microscopy methods. In this study, tetramers of the gated spinach aquaporin SoPIP2;1 were reconstituted into nanodiscs and small-angle x-ray scattering data were recorded. From these data, we refine structural models of the entire nanodisc-membrane protein complex including the flexible regions using newly developed models based on Fast Debye sums. We introduce software for these computations available via online repositories and discuss the implications and limitations of these methods. Author summaryWhen it comes to investigating the structure and function of the proteins, a particular class of proteins are known to be cumbersome and problematic: membrane proteins that reside in the cell membrane and regulate and facilitate a number of critical biological processes. Such proteins can often not be studied by conventional means as they unravel and denature structurally or even precipitate in solution. To add insult to injury, such membrane proteins also often contain parts that are intrinsically disordered rendering them irresolvable by e.g. traditional crystallographic techniques and hard to describe structurally. Here, we present a combined computational and experimental approach (as well as the necessary software) to analyze and determine the structure of such proteins in close-to-native conditions in so-called nanodiscs, a biological carrier systems, using small-angle scattering and molecular simulations.

Adiposomes are phospholipid coated triacylglyceride particles that serve as structural models of the fat storage compartments of cells, known as lipid droplets (LDs); however, unlike LDs, they do not carry proteins. There is a deficit of available methods and experimental data regarding the internal packing of the adiposomes, and computer simulations offer a promising way to pinpoint the molecular arrangements within these structures. However, in the absence of a triacylglycerol-specific atomic forcefield, thus far, all adiposome/LD simulations have been performed with the coarse grained/united atom forcefields. Yet it is desirable to model the phospholipid/triacylglycerol interface with atomic resolution. In the present study, we first prepared a 2-monooleoylglycerol (MOG) forcefield which was then used to build a trioleoylglycerol (TOG) forcefield by the modular approach of the AMBER software suite. TOG bilayer membrane (2L) systems were modelled from two different initial conformations; TOG3 and TOG2:1. The simulations revealed that TOG2:1 is the most populated conformation in TOG membranes, irrespective of the starting conformation. Some other parameter optimizations were performed for TOG membranes based on which adiposome mimicking tetralayer membrane system (4L) was prepared with a TOG bilayer at core surrounded by two DOPC leaflets. The 4L membranes were stable throughout the simulations, however it was observed that a small amount of cations and water diffused from surface to the TOG core of the membrane. Based on these results a TAG-packing model was also developed. It is expected that the availability of MOG forcefield will equip future studies with a framework for molecular dynamics simulations of adiposomes/LDs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/918136v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@e79991org.highwire.dtl.DTLVardef@113291corg.highwire.dtl.DTLVardef@f02014org.highwire.dtl.DTLVardef@ca4227_HPS_FORMAT_FIGEXP M_FIG C_FIG

Endocannabinoids are a diverse family of lipid molecules, which circulate in the human body, impacting the cardiovascular and the nervous systems. Endocannabinoids can influence pain perception, appetite, stress responses, mood, memory and learning. Regulation of these lipids present a promising therapeutic avenue for numerous neurological disorders. In addition to acting as agonists to cannabinoid receptors (CBRs), endocannabinoids can also modulate the function of various ion channels and receptors independently of CBRs. This modulation of function can arise from direct binding to the channel proteins, or via changes to the lipid properties such as membrane elasticity/stiffness, curvature, or hydrophobic thickness. Here, we assess the effects of endocannabinoids on membrane properties by examining changes in gramicidin (gA) currents in Xenopus oocytes. Endocannabinoids from both classes (Fatty acid ethanolamides (FAEs) and 2-monoacylglycerols (2-MGs)) are studied and current-voltage relationships are assessed. Employing gramicidin channels as molecular force probes can enable both predictive and quantitative studies on the impact of bilayer-mediated regulation on membrane protein function by endocannabinoids.

We use molecular dynamics simulations to characterise the local lipid annulus, or "fingerprint", of three SLC6 transporters (dDAT, hSERT, and GlyT2) embedded into a complex neuronal membrane. New membrane analysis tools were created to improve leaflet detection and leaflet-dependent properties. Overall, lipid fingerprints are comprised of similar lipids when grouped by headgroup or tail saturation. The enrichment and depletion of specific lipids, including sites of cholesterol contacts, varies between transporters. The subtle differences in lipid fingerprints results in varying membrane biophysical properties near the transporter. Through comparisons to previous literature, we highlight that the lipid-fingerprint in complex membranes is highly dependent on membrane composition. Furthermore, through embedding these transporters in a simplified model membrane, we show that the simplified membrane is not able to capture the biophysical properties of the complex membrane. Our results further characterise how the presence and identity of membrane proteins affects the complex interplay of lipid-protein interactions, including the local lipid environment and membrane biophysical properties. HIGHLIGHTSO_LILipid fingerprints are comprised of similar lipid classes C_LIO_LISites of specific lipid contacts, including CHOL, varies between transporters C_LIO_LIChanges in lipid annulus result in variable local membrane biophysical properties C_LIO_LIMembrane composition, including that of complex membranes, affects lipid annulus C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/427530v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@ae6ee9org.highwire.dtl.DTLVardef@1f39af0org.highwire.dtl.DTLVardef@412256org.highwire.dtl.DTLVardef@355f1c_HPS_FORMAT_FIGEXP M_FIG C_FIG

The Tat Translocon directly utilizes the Proton Motive Force to transport folded proteins from the n-side to the p-side of energized membranes, targeting the thylakoid lumen of chloroplasts and the periplasmic space of Bacteria and Archaea. In most organisms the Translocon consists of three subunits, TatA, TatB and TatC exhibiting a stoichiometry of [~]20-50/1/1. While TatB/TatC recognize the canonical twin-arginine motif-containing signal sequence of substrate proteins, TatA has been hypothesized to interact with TatB/TatC and translocon substrates facilitating their transport across the membrane. TatA from E.coli contains a short transmembrane helix near the N-terminus, a longer amphipathic helix and a relatively large unstructured C-terminal domain. While the transmembrane and amphipathic helixes are required for Translocon activity, the C-terminal domain is, in large measure, dispensable. TatA has been hypothesized to form higher-order oligomers in the biological membranes. In this communication we have used 1000 ns-long course-grained molecular dynamic simulations to examine the interactions between a membrane-associated E. coli TatA nonamer, alone, and in association with two Tat Translocon substrate proteins, either OEE17 or TorA. In all simulations, either in the presence or absence of substrate, the TatA nonamer markedly thinned the lipid bilayer which may facilitate substrate translocation. The pore of the nonamer was occupied by a phospholipid layer consisting of [~]6 phospholipids in the absence of substrates and [~]11 phospholipids in their presence. Structurally, the amphipathic helix of TatA were observed to exhibit significant conformational flexibility which appears to facilitate TatA-substrate interactions. In the absence of substrate the TatA nonamer was unstable with its radial architecture collapsing in 200-300 ns. In the presence of substrate, however, the radial geometry of the nonamer persists for at least 1000 ns. Interestingly, in the presence of the smaller substrate OEE17, fewer TatA monomers are retained in a radial geometry then observed in the presence of the larger substrate TorA indicating that the molecularity of the TatA oligomer can adjust to the size of the substrate. Specific hydrophilic residues of the TatA amphipathic helixes were found to interact with both substrate molecules, and these form quite stable charge-pair or hydrogen-bonding interactions. While the substrate proteins were initially placed adjacent to the amphipathic helixes of the nonamer, during the simulation trajectories the substrates moved to a more central position adjacent to, and partially entering, the oligomer pore. Concomitantly, the oligomer was observed to lose phospholipids. These latter observations may constitute a glimpse of the initial stages of protein translocation.

We have conducted all atom molecular dynamics simulations of POPC and DPPC lipid bilayers using AMBER Lipid21 force field with eight different water models, including SPC/E, TIP3P, TIP3P-FB, TIP4P-FB, TIP4P-Ew, TIP4P/2005, TIP4P-D, and OPC, to identify the most compatible one without any modification. A number of parameters have been computed in order to understand the structure of the lipid bilayer: Area per lipid, Isothermal compressibility modulus, average Volume per lipid, electron density profile, bilayer thickness, X-ray and neutron scattering form factors, deuterium order parameter, and radial distribution function. The estimated Area per lipid, Isothermal compressibility factor, volume per lipid and bilayer thickness are highly consistent with experimental results for the SPC/E water model, indicating its suitability with the AMBER Lipid21 force field, insted of any modification. The bilayer electron density profiles of both the lipid bilayers demonstrate a little augmentation of water penetration with respect to the membrane surface for TIP4P-D water model. However, the experimental X-ray and neutron scattering form factors are aligning well with the simulated results for all studied water models, and TIP4P-D shows better for X-ray data. The deuterium order parameter for lipid acyl chains value less than 0.25 for all observed water models, depicting their disorderness for both the lipid bilayers. The lateral diffusion and reorientation autocorrelation function of the lipid molecules in both the bilayers are computed to reveal their dynamics across all water models. In comparison to other water models, the simulated trajectories predict better structure and reasonably fair dynamic properties for the SPC/E water model. The TIP4P-Ew water model reproduces the lateral diffusion co-efficient in close agreement with experiment. Reorientational dynamics for both the lipids in the bilayers for eight different water models are observed; the presence of slow and slowest time components corresponds to the lipid axial motion (wobble motion) and Twist/Splay motions. So, in view of the overall performance of the different water models with the AMBER Lipid21 all atom force field in reproducing membrane physical properties, the SPC/E water model appears to be an optimal choice.

Previous molecular dynamics (MD) simulations of caveolin-1 (Cav1) in our labs revealed the possibility of two stable conformations of its intramembrane helices (H1 and H2). To distinguish between these two conformations, experimental intramolecular distances obtained using FRET (Forster resonance energy transfer) were integrated into the MD simulations to better define the position of these helices. Two mutants of Cav1 were generated where an acceptor fluorophore, dansyl, was positioned at the N-terminal end and center of H1 (A87C, F99C respectively), while a donor fluorophore, a native tryptophan, was positioned at the C-terminal end of H2 (W128). For the A87C W128 mutant, a distance of 22.5 {+/-} 0.3 [A] was observed while a distance of 24.4 {+/-} 0.2 [A] was observed for the F99C W128 mutant in phospholipid bicelles. These experimental FRET distances were compared to distances in MD simulations of over 100 Cav1 structures. Together these studies support that H1 and H2 adopt a hairpin conformation in bicelles.

Heterologous overexpression of any protein, and especially of the large transmembrane channel Nav1.5, could be associated with the insufficiency of endoplasmic reticulum folding machinery, hence leading to aspecific protein aggregation indistinguishable from the genuine --subunit interactions. In this study, we show that the interactions between heterologous Nav1.5 proteins depend on nascent N-linked glycosylation, are supported by non-native intermolecular disulfide bonds, and are likely predisposed to hydrophobic "stickiness". Particularly, we show strong interactions between the full-length Nav1.5 and its truncated peptides: N-terminal domain, all four transmembrane domains, as well as the intracellular linker between domains I and II. Taken together, we conclude that the heterologous expression system is not optimal for the identification of --subunit interaction sites of Nav1.5, and this question needs to be further addressed in the native tissues. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/679760v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@b29b7dorg.highwire.dtl.DTLVardef@1fe5909org.highwire.dtl.DTLVardef@1877990org.highwire.dtl.DTLVardef@13e04b3_HPS_FORMAT_FIGEXP M_FIG C_FIG

In protein-membrane interactions membranes provide an environment that enables proteins to fulfill multiple functions. Yet our knowledge about specificity of protein-membrane interactions is not sufficient. It is our hipothesis that this specificity is governed to a large extend by the properties of membrane itself. This study investigates the protein-membrane interactions in the case of -synuclein (S) and ceramide-1-phosphate (C1P) enriched membranes. The interactions between S and lipids were reported to be governed by various factors, including lipid composition, membrane curvature, charge and fluidity. On the other hand, C1P is anionic lipid and the shape of its molecule is similar to that of PA, which was reported to strongly interact with S. There are three main aspect of this work. First of all interactions between S and C1P enriched membranes is investigated using microscale thermophoresis. Secondly, systematic characterization of membrane systems is performed using both in vitro and in silico techniques. Eventually, principal component analysis and multiple linear regression are used to determine phenomenological dependency of those interactions in function of membrane properties.

The study focuses on investigating the interaction of SARS-CoV-2 fusion peptide fragment with model membranes of various lipid composition to elucidate the molecular mechanisms of peptide-derived membrane fusion. The work utilized the short fragment of SARS-CoV-2 fusion peptide which is homologous to 816-827 region of the native SARS-CoV-2 FP (FP816-827) and contains the highly conserved LLF motif responsible for membrane fusion, and its ineffective analogue (mFP816-827), where LLF motif was replaced for AAA. Using fluorescence fusion assay, it was demonstrated that the LLF motif plays a key role in inducing liposome fusion, whereas its replacement completely abolishes this capability. The fusogenic activity of the peptide strictly depended on the vesicle lipid composition. It was potentiated by phosphatidylethanolamine and inhibited by phosphatidylserine. Molecular dynamics revealed that both peptides predominantly adopt an -helical conformation; however, the native peptide interacts more strongly with the hydrophobic core of the membrane by increasing peptide-lipid hydrophobic contacts, while the mutant version exhibits a more superficial localization. Differential scanning microcalorimetry data indicated that the ability of FP816- 827 to disturb lipid packing increased with decreasing membrane lipid tail length. The molecular mechanisms underlying the fusogenic activity of the SARS-CoV-2 fusion peptide were identified, specifically its ability to cluster phospholipid head groups in its own vicinity. As a result, local regions with positive spontaneous curvature are formed in the outer monolayer, facilitating membrane fusion. The findings highlight the role of membrane composition and lipid architecture in the mechanism of viral fusion with host cells.