Peripheral membrane proteins (PMPs) are a subgroup of membrane-associated proteins that are water-soluble and bind to membranes, often reversibly, to perform their function. These proteins have been extensively studied in the aqueous state, but there is often a lack of high-resolution structural and functional studies of these proteins in the membrane-bound state. Currently, nuclear magnetic resonance (NMR) is among the best-equipped methods to study these relatively small proteins and domains, but current models have some disadvantages that prevent a full understanding of PMP interactions with membranes and lipids. Micelles, bicelles, and nanodiscs are all available for NMR observation but are based on synthetic lipids that may destabilize proteins or are too large to accommodate straightforward structural analysis. This protocol introduces a method for forming reverse micelles using lipids from natural sources, here called native reverse micelles. This technique allows the PMPs to embed within a shell of naturally derived lipids surrounding a small water core solubilized in an alkane solvent. PMP embedment in the lipid shell mimics binding to a cellular membrane. Here, naturally derived lipids from soy, bovine heart, and porcine brain are used in conjunction with n-dodecylphosphocholine (DPC) to encapsulate a PMP from either concentrated or dried protein, resulting in reverse micelles that may be confirmed via dynamic light scattering and NMR. This protocol allows for high-quality NMR data of PMPs interacting with membrane lipids within a biologically accurate environment. Key features • This protocol describes using natural lipids to construct reverse micelles for high-resolution NMR studies of proteins. • Initial optimization of encapsulation conditions proceeds through visual assessment, with dynamic light scattering (DLS) to measure size distribution, and NMR to observe protein behavior. • Membrane-interacting proteins are encapsulated in their membrane-bound state. Proteins that do not interact with membranes are housed in their water-solubilized state. • Structural, functional, and inhibitory studies may be performed on native reverse micelle-encapsulated proteins.

The mechanism of chemotherapeutic action of Ru-based drugs involves plasma membrane disruption and valuable insights into this process may be gained using cell membrane models. The interactions of a series of cytotoxic η6-p-cymene ruthenium(II) complexes, [Ru(η6-p-cymene)P(3,5-C(CH3)3-C6H3)3Cl2] (1), [Ru(η6-p-cymene)P(3,5-CH3-C6H3)3Cl2] (2), [Ru(η6-p-cymene)P(4-CH3O-3,5-CH3-C6H2)3Cl2] (3), and [Ru(η6-p-cymene)P(4-CH3O-C6H4)3Cl2] (4), were examined using Langmuir monolayers as simplified healthy and cancerous outer leaflet plasma membrane models. The cancerous membrane (CM1 and CM2) models contained either 40 % 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 30 % cholesterol (Chol), 20 % 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), and 10 % 1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine (DPPS). Meanwhile, the healthy membrane (HM1 and HM2) models were composed of 60 % DPPC or DOPC, 30 % Chol and 10 % DPPE. The complexes affected surface pressure isotherms and decreased compressional moduli of cancerous and healthy membrane models, interacting with the monolayers headgroup and tails according to data from polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). However, the effects did not correlate with the toxicity of the complexes to cancerous and healthy cells. Multidimensional projection technique showed that the complex (1) induced significant changes in the CM1 and HM1 monolayers, though it had the lowest cytotoxicity against cancer cells and is not toxic to healthy cells. Moreover, the most toxic complexes (2) and (4) were those that least affected CM2 and HM2 monolayers. The findings here support that the ruthenium complexes interact with lipids and cholesterol in cell membrane models, and their cytotoxic activities involve a multifaceted mode of action beyond membrane disruption.

The potentially toxic effects of emerging pollutant mixtures often deviate from the individual compound effects, presenting additive, synergistic, or agonistic interactions. This study delves into the complex world of emerging pollutants\' mixtures, with a particular focus on their potential impact on unsaturated lipid DOPC (1,2-dioleoyl-sn-glycerol-3-phosphocholine) structured as both monolayers and bilayers, which are valuable tools for mimicking cell membranes. Specifically, we examine the effects of two common types of pollutants: antibiotics (amoxicillin) and dyes (methylene blue). Utilizing Langmuir monolayers, our research reveals a synergistic effect within the pollutant mixture, as evidenced by pressure-area isotherms and polarization-modulated infrared reflection absorption spectroscopy. We identify the specific chemical interactions contributing to this synergistic effect. Furthermore, through contrast phase microscopy experiments on giant unilamellar vesicles (bilayer system), we find that the individual pollutants and the mixture exhibit similar molecular effects on the bilayer, revealing that the molecular size is a key factor in the bilayer-mixture of pollutant interaction. This highlights the importance of considering molecular size in the interactions with bilayer systems. In summary, our research dissects the critical factors of chemical interactions and molecular size concerning the effects of pollutants on DOPC, serving as simplified models of cell membranes. This study underscores the significance of comprehending the molecular effects of emerging pollutants on human health and the development of models for exploring their intricate interactions with cell membranes.

Advancing the study of membrane associated proteins and their interactions is dependent on accurate membrane models. While a variety of membrane models for high-resolution membrane protein study exist, most do not reflect the diversity of lipids found within biological membranes. In this work, we have developed native reverse micelles (nRMs) formulated with lipids from multiple eukaryotic sources, which encapsulate proteins and enable them to interact as they would with a biological membrane. Diverse formulations of nRMs using soy lecithin, porcine brain lipids, or bovine heart lipids combined with n-dodecylphosphocholine were developed and characterized by dynamic light scattering and 31 P-NMR. To optimize protein encapsulation, ubiquitin was used as a standard and protein NMR verified minimal changes to its structure. Peripheral membrane proteins, which bind reversibly to membranes, were encapsulated and include glutathione peroxidase 4 (GPx4), phosphatidylethanolamine-binding protein 1 (PEBP1), and fatty acid binding protein 4 (FABP4). All three proteins showed anticipated interactions with the membrane-like inner surface of the nRMs as assessed by protein NMR. The nRM formulations developed here allow for efficient, high-resolution study of membrane interacting proteins up to and beyond ~21 kDa, in a more biologically relevant context compared to other non-native membrane models. The approach outlined here may be applied to a wide range of lipid extracts, allowing study of a variety of membrane associated proteins in their specific biological context.

Dexamethasone (Dex) and Dexamethasone phosphate (Dex-P) are synthetic glucocorticoids with high anti-inflammatory and immunosuppressive actions that gained visibility because they reduce the mortality in critical patients with COVID-19 connected to assisted breathing. They have been widely used for the treatment of several diseases and in patients under chronic treatments, thus, it is important to understand their interaction with membranes, the first barrier when these drugs get into the body. Here, the effect of Dex and Dex-P on dimyiristoylphophatidylcholine (DMPC) membranes were studied using Langmuir films and vesicles. Our results indicate that the presence of Dex in DMPC monolayers makes them more compressible and less reflective, induces the appearance of aggregates, and suppresses the Liquid Expanded/Liquid Condensed (LE/LC) phase transition. The phosphorylated drug, Dex-P, also induces the formation of aggregates in DMPC/Dex-P films, but without disturbing the LE/LC phase transition and reflectivity. Insertion experiments demonstrate that Dex induces larger changes in surface pressure than Dex-P, due to its higher hydrophobic character. Both drugs can penetrate membranes at high lipid packings. Vesicle shape fluctuation analysis shows that Dex-P adsorption on GUVs of DMPC decreases membrane deformability. In conclusion, both drugs can penetrate and alter the mechanical properties of DMPC membranes.

Biseugenol (1), a neolignan with antiprotozoal activity against Trypanosoma cruzi, was partially methylated, and the compound obtained - methyl biseugenol (2) - had its activity evaluated against the extracellular (trypomastigotes) and intracellular (amastigotes) forms of T. cruzi. It was observed that both compounds 1 and 2 exhibited similar effects against trypomastigotes (IC50 of 11.7 and 16.2 μM, respectively), whereas compound 2 displayed higher activity against amastigotes (IC50 = 8.2 μM) in comparison with biseugenol (IC50 = 15.4 μM). Additionally, reduced toxicity against NCTC cells for compound 2 was observed (CC50 > 200 μM), differently from compound 1 with CC50 = 58.0 μM. Aiming to understand better the molecular mechanism of the biological action of compound 2, the prodrug was incorporated into cellular membrane models constituted of Langmuir monolayers of the lipids dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylserine (DPPS), and dipalmitoylphosphatidylglycerol (DPPG). The lipid-drug interaction was inferred through tensiometry, surface potential, infrared spectroscopy (PM-IRRAS), and Brewster angle microscopy (BAM). The prodrug expanded DPPC and DPPG monolayers and condensed DPPE ones, as well as presented characteristic behaviors regarding the chemical structure of the lipid considering expansion-compression curves, surface potential-area isotherms, and stability of previously compressed monolayers to relevant-biological surface pressures. PM-IRRAS indicated a molecular disorder for DPPC and DPPS alkyl chains in the presence of the drug. BAM revealed the presence of domains in the DPPG and DPPE monolayers, which was probably induced by the prodrug. These data suggest, in general, that the lipid composition modulates the interaction of compound 2, whose results are expected to correlate to its trypanocidal activity, which involves the plasma membrane of T. cruzi as the primary target, i.e., the first barrier that the compound should encounter to interact with the microorganism.

It has been a long journey from Pliny the Elder (23-79 AD) to the FDA approval of the first injectable nanomedicine in 1997. A journey powered by intellectual curiosity, which began with sprinkling olive oil on seawater and culminated in playing around with smears of egg lecithin on microscopic slides. This brief review highlights how a few pairs of gifted hands attached to highly motivated brains have turned a curious discovery made under a microscopic lens into novel nanotherapeutics including liposome-based anti-cancer drugs and potent liposomal vaccines given to millions.

The Fluid-Mosaic Membrane (FMM) model was originally proposed as a general, nanometer-scale representation of cell membranes (Singer and Nicolson, 1972). The FMM model was based on some general principles, such as thermodynamic considerations, intercalation of globular proteins into a lipid bilayer, independent protein and lipid dynamics, cooperativity and other characteristics. Other models had trimolecular structures or membrane globular lipoprotein units. These latter models were flawed, because they did not allow autonomous lipids, membrane domains or discrete lateral dynamics. The FMM model was also consistent with membrane asymmetry, cis- and trans-membrane linkages and associations of membrane components into multi-molecular complexes and domains. It has remained useful for explaining the basic organizational principles and properties of various biological membranes. New information has been added, such as membrane-associated cytoskeletal assemblies, extracellular matrix interactions, transmembrane controls, specialized lipid-protein domains that differ in compositions, rotational and lateral mobilities, lifetimes, functions, and other characteristics. The presence of dense, structured membrane domains has reduced significantly the extent of fluid-lipid membrane areas, and the FMM model is now considered to be more mosaic and dense than the original proposal.

Among pollutants of emerging concern, endocrine disruptors (ED) have been shown to cause side effects in humans and animals. Bisphenol-A (BPA) is an ED by-product of the plastic industry and one of the chemicals with the highest volume produced yearly. Here, we studied the role of cholesterol in the BPA exposure effects over membrane models. We used Langmuir films of both neat lipid DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and cholesterol (Chol) and a binary mixture containing DPPC/Chol, exposing it to BPA. We evaluate changes in the π-A isotherms and the PM-IRRAS (polarization modulation-infrared reflection adsorption spectroscopy) spectra. BPA exposure induced changes in the DPPC and Chol neat monolayers, causing mean molecular area expansion and altering profiles. However, at high surface pressure, the BPA was expelled from the air-water interface. For the DPPC/Chol mixture, BPA caused expansion throughout the whole compression, indicating that BPA is present at the monolayer interface. The PM-IRRAS analysis showed that BPA interacted with the phosphate group of DPPC through hydrogen bonding, which caused the area\'s expansion. Such evidence might be biologically relevant to better understand the mechanism of action of BPA in cell membranes once phosphatidylcholines and Chol are found in mammalian membranes.

Plants have to constantly face pathogen attacks. To cope with diseases, they have to detect the invading pathogen as early as possible via the sensing of conserved motifs called invasion patterns. The first step of perception occurs at the plasma membrane. While many invasion patterns are perceived by specific proteinaceous immune receptors, several studies have highlighted the influence of the lipid composition and dynamics of the plasma membrane in the sensing of invasion patterns. In this review, we summarize current knowledge on how some microbial invasion patterns could interact with the lipids of the plasma membrane, leading to a plant immune response. Depending on the invasion pattern, different mechanisms are involved. This review outlines the potential of combining biological with biophysical approaches to decipher how plasma membrane lipids are involved in the perception of microbial invasion patterns.