α-Synuclein is a presynaptic protein that binds to cell membranes and is linked to Parkinson's disease (PD). Whilst the normal function of remains α-synuclein remains uncertain, it is thought that oligomerization of the protein on the cell membrane contributes to cell damage.Knowledge of how α-synuclein binds to lipid bilayers is therefore of great interest as a likely first step in the molecular pathophysiology of PD, and may provide insight of the phenotype of PD-promoting mutations. We use coarse-grained and atomistic simulations in conjunction with NMR and cross-linking mass spectrometry studies of α-synuclein bound to anionic lipid bilayers to reveal a break in the helical structure of the NAC region, which may give rise to subsequent oligomer formation. Coarse-grained simulations of α-synuclein show that the interhelical region leads recognition and binding to both POPG and mixed composition bilayers and identifies important protein-lipid contacts, including those in the region between the two helices in the folded structure. We extend these simulations with all-atom simulations of the initial binding event to reveal details of the time-progression of lipid binding. We present secondary structure analysis that reveals points of possible β-strand formation in the structure, and investigate intramolecular contacts with simulations and mass-spectrometry crosslinking.Additionally we show how Markov state models can be used to investigate possible conformational changes of membrane bound α-synuclein in the NAC region, and we extract representative structures. These structural insights will aid the design and development of novel therapeutic approaches.a-syn_ms_v19 biorxiv.docx 17-Jun-20 α-Synuclein is a protein implicated in neurodegenerative disorders including Parkinson's disease and Lewy body dementia (1). Its function in healthy neurons remains uncertain (2).Lipid bilayer association of α-synuclein is thought to be important for its biological function in regulating synaptic vesicles, where it has been shown to be essential for SNARE complex assembly at the presynaptic membrane (3,4). It has been postulated to have a wide range of functions, including neuronal differentiation (4) and suppression of apoptosis (5). It is thought that toxicity towards neurones arises from the interaction of misfolded/aggregated α-synuclein with the lipid bilayer component of cell membranes (6). Thus, defining the interactions between α-synuclein and lipid bilayers is a key step in understanding the mode of action of αsynuclein, thus helping to provide a route towards drug research aimed at presenting or reversing its cellular effects .Native α-synuclein is an intrinsically disordered monomeric protein in solution (7-9). SAXS and NMR-derived experimental data suggest relatively small amounts of compaction in the ensemble of free α-synuclein (10,11). In contrast to these observations some discrete molecular dynamics (MD) simulations (12) combined with crosslinking data (13) have been interpreted as suggesting that part of the ens...

of pneumonia, daptomycin is curiously inhibited by lung surfactant and is rendered ineffective against Gram-positive Streptococcus pneumoniae, the primary cause of this illness. The reason for this is currently not understood. We developed lipid model systems to mimic S. pneumoniae membranes, tissue cell membranes, and both synthetic and natural (BLES) lung surfactant. Using the Langmuir-Blodgett trough, constant-area insertion assays were performed. It was discovered that daptomycin inserts into lung surfactant to a greater extent than it does with bacterial membrane at physiologicallyrelevant calcium concentrations. Fluorescence spectroscopy data provided us with results corroborating the findings from the insertion assays. Meanwhile, compression isotherms provided data on monolayer compressibility. We found that daptomycin and calcium can improve surfactant activity and performance, suggesting the formation of multilayers at lower pressures. Atomic force microscopy and Kelvin probe force microscopy imaging provided visual evidence of multilayer formation induced by the presence of daptomycin and calcium, confirming this mechanism of inhibition. Based on these results, we propose a novel model of daptomycin sequestration by lung surfactant (see figure ), where (1) daptomycin inserts into surfactant, (2) lowers its compressibility, (3) induces multilayer formation, and (4) reinforces its sequestration, rendering it unable to exert its bactericidal activity against S. pneumoniae.