Phospholipid Membranes as Chemically and Functionally Tunable Materials.

The sheet-like lipid bilayer is the fundamental structural component of all cell membranes. Its building blocks are phospholipids and cholesterol. Their amphiphilic structure spontaneously leads to the formation of a bilayer in aqueous environment. Lipids are not just structural elements. Individual lipid species, the lipid membrane structure, and lipid dynamics influence and regulate membrane protein function. An exciting field is emerging where the membrane-associated material properties of different bilayer systems are used in designing innovative solutions for widespread applications across various fields, such as the food industry, cosmetics, nano- and biomedicine, drug storage and delivery, biotechnology, nano- and biosensors, and computing. Here, the authors summarize what is known about how lipids determine the properties and functions of biological membranes and how this has been or can be translated into innovative applications. Based on recent progress in the understanding of membrane structure, dynamics, and physical properties, a perspective is provided on how membrane-controlled regulation of protein functions can extend current applications and even offer new applications.

Conformation of Pyroglutamated Amyloid ß (3-40) and (11-40) Fibrils - Extended or Hairpin?

Amyloid ß (Aß) is a hallmark protein of Alzheimer's disease. One physiologically important Aß variant is formed by initial N-terminal truncation at a glutamic acid position (either E3 or E11), which is subsequently cyclized to a pyroglutamate (either pE3 or pE11). Both forms have been found in high concentrations in the core of amyloid plaques and are likely of high importance in the pathology of Alzheimer's disease. However, the molecular structure of the fibrils of these variants is not entirely clear. Solid-state NMR spectroscopy studies have reported a molecular contact between Gly25 and Ile31, which would disagree with the conventional hairpin model of wildtype (WT-)Aß1-40 fibrils, most often described in the literature. We investigated the conformation of the monomeric unit of pE3-Aß3-40 and pE11-Aß11-40 (and for comparison also wildtype (WT)-Aß1-40) fibrils to find out whether the hairpin or a newly suggested extended structure dominates the structure of the Aß monomers in these fibrils. To this end, solid-state NMR spectroscopy was applied probing the inter-residual contacts between Phe19/Leu34, Ala21/Leu34, and especially Gly25/Ile31 using suitable isotopic labeling schemes. In the second part, the flexible turn of the Aß40 peptides was replaced by a (3-(3-aminomethyl)phenylazo)phenylacetic acid (AMPP)-based photoswitch, which can predefine the peptide conformation to either an extended (trans) or hairpin (cis) conformation. This enables simultaneous spectroscopic assessment of the conformation of the AMPP-photoswitch, allowing in situ structural investigations during fibrillation in contrast to structural techniques such as NMR spectroscopy or cryo-EM, which can only be applied to stable conformers. Both methods confirm an extended structure for the peptidic monomers in fibrils of all investigated Aß variants. Especially the Gly25/Ile31 contact is a decisive indicator for the extended structure along with the characteristic absorption spectra of trans-AMPP-Aß.

Membrane-Mediated Allosteric Action of Serotonin on a Noncognate G-Protein-Coupled Receptor.

The structure and dynamics of the lipid membrane can affect the activity of membrane proteins. Therefore, small lipophilic molecules that alter membrane properties (such as the neurotransmitter serotonin) can potentially modulate receptor activity without binding to the receptor. Here, we investigated how the activity of neuropeptide Y type 4 receptor (Y4R, reconstituted in lipid bicelles) is modulated by serotonin, which has no known interaction with Y4R. We found a serotonin-concentration-dependent decrease (down to 0.1 mM of serotonin) in the ligand affinity of Y4R. This effect correlates with a serotonin-induced reduction of the resistance of the bilayer to indentation (measured by atomic force microscopy) and bilayer thickness (measured by solid state NMR) in two different types of zwitterionic lipid bicelles. Our findings indicate a "membrane-mediated allosteric effect" of serotonin on the activation of Y4R and suggest the potential for developing pharmacophores, which can modulate cellular signaling without directly interacting with any receptor.

Effects of imidazolium ionic liquids on skin barrier lipids - Perspectives for drug delivery.

Ionic liquids (ILs) have great potential to facilitate transdermal and topical drug delivery. Here, we investigated the mechanism of action of amphiphilic ILs 1-methyl-3-octylimidazolium bromide (C8MIM) and 3-dodecyl-1-methylimidazolium bromide (C12MIM) in skin barrier lipid models in comparison to their complex effects in human skin. C8MIM incorporated in a skin lipid model was a better permeation enhancer than C12MIM for water and model drugs, theophylline and diclofenac. Solid state 2H NMR and X-ray diffraction indicated that both ILs prefer the cholesterol-rich regions in skin lipids without significantly perturbing their lamellar arrangement and that C8MIM induces the formation of an isotropic lipid phase to a greater extent compared to C12MIM. C12MIM applied topically to the lipid model or human skin as a pretreatment was more potent than C8MIM. When co-applied with the drugs to human skin, aqueous C12MIM was more potent than C8MIM in enhancing theophylline permeation, but neither IL affected (even decreased) diclofenac permeation. Thus, the IL's ability to permeabilize skin lipid barrier is strongly modulated by its ability to reach the site of action and its interactions with drug and solvent. Such an interplay is far from trivial and requires detailed investigation to realize the full potential of ILs.

Detecting Protein-Ligand Interactions with Nitroxide Based Paramagnetic Cosolutes.

NMR spectroscopy techniques can provide important information about protein-ligand interactions. Here we tested an NMR approach which relies on the measurement of paramagnetic relaxation enhancements (PREs) arising from analogous cationic, anionic or neutral soluble nitroxide molecules, which distribute around the protein-ligand complex depending on near-surface electrostatic potentials. We applied this approach to two protein-ligand systems, interleukin-8 interacting with highly charged glycosaminoglycans and the SH2 domain of Grb2 interacting with less charged phospho-tyrosine tripeptides. The electrostatic potential around interleukin-8 and its changes upon binding of glycosaminoglycans could be derived from the PRE data and confirmed by theoretical predictions from Poisson-Boltzmann calculations. The ligand influence on the PREs and NMR-derived electrostatic potentials of Grb2 SH2 was localized to a narrow protein region which allowed the localization of the peptide binding pocket. Our analysis suggests that experiments with nitroxide cosolutes can be useful for investigating protein-ligand electrostatic interactions and mapping ligand binding sites.

Dimerization and Crowding in the Binding of Interleukin 8 to Dendritic Glycosaminoglycans as Artificial Proteoglycans.

The interactions of glycosaminoglycans (GAG) with proteins of the extracellular matrix govern and regulate complex physiological functions including cellular growth, immune response, and inflammation. Repetitive presentation of GAG binding motifs, as found in native proteoglycans, might enhance GAG-protein binding through multivalent interactions. Here, we report the chemical synthesis of dendritic GAG oligomers constructed of nonasulfated hyaluronan tetrasaccharides for investigating the binding of the protein chemokine interleukin 8 (IL-8) to artificial, well-defined proteoglycan architectures. Binding of mutant monomeric and native dimerizable IL-8 was investigated by NMR spectroscopy and isothermal titration calorimetry. Dendritic oligomerization of GAG increased the binding affinity of both monomeric and dimeric IL-8. Monomeric IL-8 bound to monomeric and dimeric GAG with KD values of 7.3 and 0.108 µM, respectively. The effect was less pronounced for dimerizable wild-type IL-8, for which GAG dimerization improved the affinity from 34 to 5 nM. Binding of dimeric IL-8 to oligomeric GAG was limited by steric crowding effects, strongly reducing the affinity of subsequent binding events. In conclusion, the strongest effect of GAG oligomerization was the amplified binding of IL-8 monomers, which might concentrate monomeric protein in the extracellular matrix and thus promote protein dimerization under physiological conditions.

Ready for the sheet: ß-strand folding of phosphorylation clusters guides GPCR binding to arrestin.

The molecular dynamics of arrestin binding to G protein-coupled receptors (GPCRs) are still poorly understood. In this issue of Structure, Guillien et al. show that negative charges in GPCR key phosphorylation clusters induce the formation of a transient ß-strand that participates in an intermolecular ß-sheet in the associated complex.

Myr-Arf1 conformational flexibility at the membrane surface sheds light on the interactions with ArfGAP ASAP1.

ADP-ribosylation factor 1 (Arf1) interacts with multiple cellular partners and membranes to regulate intracellular traffic, organelle structure and actin dynamics. Defining the dynamic conformational landscape of Arf1 in its active form, when bound to the membrane, is of high functional relevance and key to understanding how Arf1 can alter diverse cellular processes. Through concerted application of nuclear magnetic resonance (NMR), neutron reflectometry (NR) and molecular dynamics (MD) simulations, we show that, while Arf1 is anchored to the membrane through its N-terminal myristoylated amphipathic helix, the G domain explores a large conformational space, existing in a dynamic equilibrium between membrane-associated and membrane-distal conformations. These configurational dynamics expose different interfaces for interaction with effectors. Interaction with the Pleckstrin homology domain of ASAP1, an Arf-GTPase activating protein (ArfGAP), restricts motions of the G domain to lock it in what seems to be a conformation exposing functionally relevant regions.

Nuclear magnetic resonance spectroscopy to quantify major extracellular matrix components in fibro-calcific aortic valve disease.

Fibro-calcific aortic valve disease (FCAVD) is a pathological condition marked by overt fibrous and calcific extracellular matrix (ECM) accumulation that leads to valvular dysfunction and left ventricular outflow obstruction. Costly valve implantation is the only approved therapy. Multiple pharmacological interventions are under clinical investigation, however, none has proven clinically beneficial. This failure of translational approaches indicates incomplete understanding of the underlying pathomechanisms and may result from a limited toolbox of scientific methods to assess the cornerstones of FCAVD: lipid deposition, fibrous and calcific ECM accumulation. In this study, we evaluated magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy to both, qualitatively and quantitatively assess these key elements of FCAVD pathogenesis. NMR spectra showed collagen, elastin, triacylglycerols, and phospholipids in human control and FCAVD tissue samples (n = 5). Calcification, measured by the hydroxyapatite content, was detectable in FCAVD tissues and in valve interstitial cells under procalcifying media conditions. Hydroxyapatite was significantly higher in FCAVD tissues than in controls (p < 0.05) as measured by 31P MAS NMR. The relative collagen content was lower in FCAVD tissues vs. controls (p < 0.05). Overall, we demonstrate the versatility of NMR spectroscopy as a diagnostic tool in preclinical FCAVD assessment.

Ligand binding of interleukin-8: a comparison of glycosaminoglycans and acidic peptides.

Recognition and binding of regulatory proteins to glycosaminoglycans (GAGs) from the extracellular matrix is a process of high biological importance. The interaction between negatively charged sulfate or carboxyl groups of the GAGs and clusters of basic amino acids on the protein is crucial in this binding process and it is believed that electrostatics represent the key factor for this interaction. However, given the rather undirected nature of electrostatics, it is important to achieve a clear understanding of its role in protein-GAG interactions and how specificity and selectivity in these systems can be achieved, when the classical key-lock binding motif is not applicable. Here, we compare protein binding of a highly charged heparin (HP) hexasaccharide with four de novo designed decapeptides of varying negative net charge. The charge density of these peptides was comparable to typical GAGs of the extracellular matrix. We used the regulatory protein interleukin-8 (IL-8) because its interactions with GAGs are well described. All four peptide ligands bind to the same epitope of IL-8 but show much weaker binding affinity as revealed in 1H-15N HSQC NMR titration experiments. Complementary molecular docking and molecular dynamics simulations revealed further atomistic details of the interaction mode of GAG versus peptide ligands. Overall, similar contributions to the binding energy and hydrogen bond formation are determined for HP and the highly charged peptides, suggesting that the entropic loss of the peptides upon binding likely account for the remarkably different affinity of GAG versus peptide ligands to IL-8.

Conformational State of Fenamates at the Membrane Interface: A MAS NOESY Study.

The present work analyzes the 1H NOESY MAS NMR spectra of three fenamates (mefenamic, tolfenamic, and flufenamic acids) localized in the lipid-water interface of phosphatidyloleoylphosphatidylcholine (POPC) membranes. The observed cross-peaks in the two-dimensional NMR spectra characterized intramolecular proximities between the hydrogen atoms of the fenamates as well as intermolecular interactions between the fenamates and POPC molecules. The peak amplitude normalization for an improved cross-relaxation (PANIC) approach, the isolated spin-pair approximation (ISPA) model, and the two-position exchange model were used to calculate the interproton distances indicative of specific conformations of the fenamates. The results showed that the proportions of the A+C and B+D conformer groups of mefenamic and tolfenamic acids in the presence of POPC were comparable within the experimental error and amounted to 47.8%/52.2% and 47.7%/52.3%, respectively. In contrast, these proportions for the flufenamic acid conformers differed and amounted to 56.6%/43.4%. This allowed us to conclude that when they bind to the POPC model lipid membrane, fenamate molecules change their conformational equilibria.

Analysis of the Dynamics of the Human Growth Hormone Secretagogue Receptor Reveals Insights into the Energy Landscape of the Molecule.

G protein-coupled receptors initiate signal transduction in response to ligand binding. Growth hormone secretagogue receptor (GHSR), the focus of this study, binds the 28 residue peptide ghrelin. While structures of GHSR in different states of activation are available, dynamics within each state have not been investigated in depth. We analyze long molecular dynamics simulation trajectories using "detectors" to compare dynamics of the apo and ghrelin-bound states yielding timescale-specific amplitudes of motion. We identify differences in dynamics between apo and ghrelin-bound GHSR in the extracellular loop 2 and transmembrane helices 5-7. NMR of the GHSR histidine residues reveals chemical shift differences in these regions. We evaluate timescale specific correlation of motions between residues of ghrelin and GHSR, where binding yields a high degree of correlation for the first 8 ghrelin residues, but less correlation for the helical end. Finally, we investigate the traverse of GHSR over a rugged energy landscape via principal component analysis.

The intriguing molecular dynamics of Cer[EOS] in rigid skin barrier lipid layers requires improvement of the model.

Omega-O-acyl ceramides such as 32-linoleoyloxydotriacontanoyl sphingosine (Cer[EOS]) are essential components of the lipid skin barrier, which protects our body from excessive water loss and the penetration of unwanted substances. These ceramides drive the lipid assembly to epidermal-specific long periodicity phase (LPP), structurally much different than conventional lipid bilayers. Here, we synthesized Cer[EOS] with selectively deuterated segments of the ultralong N-acyl chain or deuterated or 13C-labeled linoleic acid and studied their molecular behavior in a skin lipid model. Solid-state 2H NMR data revealed surprising molecular dynamics for the ultralong N-acyl chain of Cer[EOS] with increased isotropic motion toward the isotropic ester-bound linoleate. The sphingosine moiety of Cer[EOS] is also highly mobile at skin temperature, in stark contrast to the other LPP components, N-lignoceroyl sphingosine acyl, lignoceric acid, and cholesterol, which are predominantly rigid. The dynamics of the linoleic chain is quantitatively described by distributions of correlation times and using dynamic detector analysis. These NMR results along with neutron diffraction data suggest an LPP structure with alternating fluid (sphingosine chain-rich), rigid (acyl chain-rich), isotropic (linoleate-rich), rigid (acyl-chain rich), and fluid layers (sphingosine chain-rich). Such an arrangement of the skin barrier lipids with rigid layers separated with two different dynamic "fillings" i) agrees well with ultrastructural data, ii) satisfies the need for simultaneous rigidity (to ensure low permeability) and fluidity (to ensure elasticity, accommodate enzymes, or antimicrobial peptides), and iii) offers a straightforward way to remodel the lamellar body lipids into the final lipid barrier.

How Single Site Mutations Can Help Understanding Structure Formation of Amyloid ß1-40.

Amyloid fibrils represent the structural endpoint on the energetic (mis)folding landscape of very many proteins. Physiologically, amyloid fibrils are observed as a characteristic hallmark in misfolding diseases often associated with degenerative and neurodegenerative disorders. In the beginning of the scientific discussion, the focus is laid on the fibrillar state, but over the time it becomes increasingly clear that low molecular weight and transient aggregates are of crucial importance for pathological mechanisms. Structural studies find different intra- and intermolecular contacts for the most well-studied peptide amyloid ß (Aß) depending on the stage of fibrillation. In particular, the contact between residues phenylalanine 19 (F19) and leucine 34 (L34) seems to be highly conserved, suggesting that it must be of particular significance for Aß misfolding and possibly the pathological properties of the peptide. This review aims to highlight the rational and the usefulness of point mutations in Aß peptides and their impact on the critical interstrand contact F19-L34 depending on the stage of fibrillation. While the amyloid structure of Aß is very robust against quite a few modifications, the toxicity of mutated Aß molecules highly depends on the F19-L34 contact.

Unusual Robustness of Neurotransmitter Vesicle Membranes against Serotonin-Induced Perturbations.

Nature confines hundreds of millimolar of amphiphilic neurotransmitters, such as serotonin, in synaptic vesicles. This appears to be a puzzle, as the mechanical properties of lipid bilayer membranes of individual major polar lipid constituents of synaptic vesicles [phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS)] are significantly affected by serotonin, sometimes even at few millimolar concentrations. These properties are measured by atomic force microscopy, and their results are corroborated by molecular dynamics simulations. Complementary 2H solid-state NMR measurements also show that the lipid acyl chain order parameters are strongly affected by serotonin. The resolution of the puzzle lies in the remarkably different properties displayed by the mixture of these lipids, at molar ratios mimicking those of natural vesicles (PC:PE:PS:Cholesterol = 3:5:2:5). Bilayers constituting of these lipids are minimally perturbed by serotonin, and show only a graded response at physiological concentrations (>100 mM). Significantly, the cholesterol (up to 33% molar ratio) plays only a minor role in dictating these mechanical perturbations, with PC:PE:PS:Cholesterol = 3:5:2:5 and 3:5:2:0 showing similar perturbations. We infer that nature uses an emergent mechanical property of a specific mixture of lipids, all individually vulnerable to serotonin, to appropriately respond to physiological serotonin levels.

Different membrane order measurement techniques are not mutually consistent.

"Membrane order" is a term commonly used to describe the elastic and mechanical properties of the lipid bilayer, though its exact meaning is somewhat context- and method dependent. These mechanical properties of the membrane control many cellular functions and are measured using various biophysical techniques. Here, we ask if the results obtained from various techniques are mutually consistent. Such consistency cannot be assumed a priori because these techniques probe different spatial locations and different spatial and temporal scales. We evaluate the change of membrane order induced by serotonin using nine different techniques in lipid bilayers of three different compositions. Serotonin is an important neurotransmitter present at 100s of mM concentrations in neurotransmitter vesicles, and therefore its interaction with the lipid bilayer is biologically relevant. Our measurement tools include fluorescence of lipophilic dyes (Nile Red, Laurdan, TMA-DPH, DPH), whose properties are a function of membrane order; atomic force spectroscopy, which provides a measure of the force required to indent the lipid bilayer; 2H solid-state NMR spectroscopy, which measures the molecular order of the lipid acyl chain segments; fluorescence correlation spectroscopy, which provides a measure of the diffusivity of the probe in the membrane; and Raman spectroscopy, where spectral intensity ratios are affected by acyl chain order. We find that different measures often do not correlate with each other and sometimes even yield conflicting results. We conclude that no probe provides a general measure of membrane order and that any inference based on the change of membrane order measured by a particular probe may be unreliable.

Rhomboid-catalyzed intramembrane proteolysis requires hydrophobic matching with the surrounding lipid bilayer.

Membrane thinning by rhomboid proteins has been proposed to reduce hydrophobic mismatch, providing a unique environment for important functions ranging from intramembrane proteolysis to retrotranslocation in protein degradation. We show by in vitro reconstitution and solid-state nuclear magnetic resonance that the lipid environment of the Escherichia coli rhomboid protease GlpG influences its activity with an optimal hydrophobic membrane thickness between 24 and 26 Å. While phosphatidylcholine membranes are only negligibly altered by GlpG, in an E. coli-relevant lipid mix of phosphatidylethanolamine and phosphatidylglycerol, a thinning by 1.1 Å per leaflet is observed. Protease activity is strongly correlated with membrane thickness and shows no lipid headgroup specificity. We infer from these results that, by adjusting the thickness of specific membrane domains, membrane proteins shape the bilayer for their specific needs.

The effect of cholesterol on highly curved membranes measured by nanosecond Fluorescence Correlation Spectroscopy.

Small lipid vesicles (with diameter ≤100 nm) with their highly curved membranes comprise a special class of biological lipid bilayers. The mechanical properties of such membranes are critical for their function, e.g. exocytosis. Cholesterol is a near-universal regulator of membrane properties in animal cells. Yet measurements of the effect of cholesterol on the mechanical properties of membranes have remained challenging, and the interpretation of such measurements has remained a matter of debate. Here we show that nanosecond fluorescence correlation spectroscopy (FCS) can directly measure the ns-microsecond rotational correlation time (τr) of a lipid probe in high curvature vesicles with extraordinary sensitivity. Using a home-built 4-Pi fluorescence cross-correlation spectrometer containing polarization-modulating elements, we measure the rotational correlation time (τr) of Nile Red in neurotransmitter vesicle mimics. As the cholesterol mole fraction increases from 0 to 50%,τrincreases from 17 ± 1 to 112 ± 12 ns, indicating a viscosity change of nearly a factor of 7. These measurements are corroborated by solid-state NMR results, which show that the order parameter of the lipid acyl chains increases by about 50% for the same change in cholesterol concentration. Additionally, we measured the spectral parameters of polarity-sensitive fluorescence dyes, which provide an indirect measure of viscosity. The green/red ratio of Nile Red and the generalized polarization of Laurdan show consistent increases of 1.3× and 2.6×, respectively. Our results demonstrate that rotational FCS can directly measure the viscosity of highly curved membranes with higher sensitivity and a wider dynamic range compared to other conventional techniques. Significantly, we observe that the viscosity of neurotransmitter vesicle mimics is remarkably sensitive to their cholesterol content.

Mechanistic insights into the size-dependent effects of nanoparticles on inhibiting and accelerating amyloid fibril formation.

The aggregation of peptides into amyloid fibrils has been linked to ageing-related diseases, such as Alzheimer's and type 2 diabetes. Interfaces, particularly those with large nanostructured surfaces, can affect the kinetics of peptide aggregation, which ranges from complete inhibition to strong acceleration. While a number of physiochemical parameters determine interfacial effects, we focus here on the role of nanoparticle (NP) size and curvature. We used thioflavin T (ThT) fluorescence assays to demonstrate the size-dependent effects of NPs on amyloid fibril formation for the peptides Aß40, NNFGAIL, GNNQQNY and VQIYVK. While 5 nm gold NPs (AuNP-5) retarded or inhibited the aggregation of all peptides except NNFGAIL, larger 20 nm gold NPs (AuNP-20) tended to accelerate or not influence peptide aggregation. Differences in the NP effects for the peptides resulted from the different peptide properties (size, tendency to aggregate) and associated surface binding affinities. Additional dynamic light scattering (DLS), electron microscopy, and atomic force microscopy (AFM) experiments with the Aß40 peptide confirmed size-dependent NP effects on peptide aggregation, and also suggested a structural influence on the formed fibrils. NPs can serve as a surface for the adsorption of peptide monomers and enable nucleation to oligomers and fibril formation. However, molecular dynamics (MD) simulations showed that peptide oligomers were less stable at smaller NPs. High surface curvatures destabilized prefibrillar structures, which provides a possible explanation for inhibitory effects on fibril growth, provided that peptide-NP surface binding was relevant for fibril formation. These mechanistic insights can support the design of future nanostructured materials.

Serotonergic drugs modulate the phase behavior of complex lipid bilayers.

Serotonin is an endogenous neurotransmitter involved in both physiological and pathophysiological processes. Traditionally, serotonin acts as a ligand for G protein-coupled receptors (GPCRs) leading to subsequent cell signaling. However, serotonin can also bind to lipid membranes with high affinity and modulate the phase behavior in 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC)/N-palmitoyl-D-erythro-sphingosylphosphorylcholine (PSM)/cholesterol model membranes mimicking the outer leaflet of the plasma membrane. Here, we investigated if serotonergic drugs containing the pharmacophore from serotonin would also modulate phase behavior in lipid membranes in a similar fashion. We used 2H NMR spectroscopy to explore the phase behavior of POPC/PSM/cholesterol (4/4/2 molar ratio) mixtures in the presence of the serotonergic drugs aripiprazole, BRL-54443, BW-723C86, and CP-135807 at a lipid to drug molar ratio of 10:1. POPC and PSM were perdeuterated in the palmitoyl chain, respectively, and prepared in individual samples. Numerical lineshape simulations of the 2H NMR spectra were used to calculate the order parameter profiles and projected lengths of the saturated acyl chains. All serotonergic drugs induce two components in 2H NMR spectra, indicating that they increased the hydrophobic mismatch between the thickness of the coexisting lipid phases leading to larger domain sizes, relatively similarly to serotonin. AFM force indentation and Raman spectral studies, which interrogate membrane mechanical properties, also indicate changes in membrane order in the presence of these drugs. These findings highlight how serotonergic drugs alter membrane phase behavior and could modulate both target and other membrane proteins, possibly explaining the side effects observed for serotonergic and other clinically relevant drugs.