Rapid Formation of Non-canonical Phospholipid Membranes by Chemoselective Amide-Forming Ligations with Hydroxylamines.

There has been increasing interest in methods to generate synthetic lipid membranes as key constituents of artificial cells or to develop new tools for remodeling membranes in living cells. However, the biosynthesis of phospholipids involves elaborate enzymatic pathways that are challenging to reconstitute in vitro. An alternative approach is to use chemical reactions to non-enzymatically generate natural or non-canonical phospholipids de novo. Previous reports have shown that synthetic lipid membranes can be formed in situ using various ligation chemistries, but these methods lack biocompatibility and/or suffer from slow kinetics at physiological pH. Thus, it would be valuable to develop chemoselective strategies for synthesizing phospholipids from water-soluble precursors that are compatible with synthetic or living cells Here, we demonstrate that amide-forming ligations between lipid precursors bearing hydroxylamines and α-ketoacids (KAs) or potassium acyltrifluoroborates (KATs) can be used to prepare non-canonical phospholipids at physiological pH conditions. The generated amide-linked phospholipids spontaneously self-assemble into cell-like micron-sized vesicles similar to natural phospholipid membranes. We show that lipid synthesis using KAT ligation proceeds extremely rapidly, and the high selectivity and biocompatibility of the approach facilitates the in situ synthesis of phospholipids and associated membranes in living cells.

Chemoselective Esterification of Natural and Prebiotic 1,2-Amino Alcohol Amphiphiles in Water.

In cells, a vast number of membrane lipids are formed by the enzymatic O-acylation of polar head groups with acylating agents such as fatty acyl-CoAs. Although such ester-containing lipids appear to be a requirement for life on earth, it is unclear if similar types of lipids could have spontaneously formed in the absence of enzymatic machinery at the origin of life. There are few examples of enzyme-free esterification of amphiphiles in water and none that can occur in water at physiological pH using biochemically relevant acylating agents. Here we report the unexpected chemoselective O-acylation of 1,2-amino alcohol amphiphiles in water directed by Cu(II) and several other transition metal ions. In buffers containing Cu(II) ions, mixing biological 1,2-amino alcohol amphiphiles such as sphingosylphosphorylcholine with biochemically relevant acylating agents, namely, acyl adenylates and acyl-CoAs, leads to the formation of the O-acylation product with high selectivity. The resulting O-acylated sphingolipids self-assemble into vesicles with markedly different biophysical properties than those formed from their N-acyl counterparts. We also demonstrate that Cu(II) can direct the O-acylation of alternative 1,2-amino alcohols, including prebiotically relevant 1,2-amino alcohol amphiphiles, suggesting that simple mechanisms for aqueous esterification may have been prevalent on earth before the evolution of enzymes.

Surfactant-Mediated Structural Modulations to Planar, Amphiphilic Multilamellar Stacks.

The hydrophobic effect, a ubiquitous process in biology, is a primary thermodynamic driver of amphiphilic self-assembly. It leads to the formation of unique morphologies including two highly important classes of lamellar and micellar mesophases. The interactions between these two types of structures and their involved components have garnered significant interest because of their importance in key biochemical technologies related to the isolation, purification, and reconstitution of membrane proteins. This work investigates the structural organization of mixtures of the lamellar-forming phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and two zwitterionic micelle-forming surfactants, being n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (Zwittergent 3-12 or DDAPS) and 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (O-Lyso-PC), when assembled by water vapor hydration with X-ray diffraction measurements, brightfield optical microscopy, wide-field fluorescence microscopy, and atomic force microscopy. The results reveal that multilamellar mesophases of these mixtures can be assembled across a wide range of POPC to surfactant (POPC:surfactant) concentration ratios, including ratios far surpassing the classical detergent-saturation limit of POPC bilayers without significant morphological disruptions to the lamellar motif. The mixed mesophases generally decreased in lamellar spacing (D) and headgroup-to-headgroup distance (Dhh) with a higher concentration of the doped surfactant, but trends in water layer thickness (Dw) between each bilayer in the stack are highly variable. Further structural characteristics including mesophase topography, bilayer thickness, and lamellar rupture force were revealed by atomic force microscopy (AFM), exhibiting homogeneous multilamellar stacks with no significant physical differences with changes in the surfactant concentration within the mesophases. Taken together, the outcomes present the assembly of unanticipated and highly unique mixed mesophases with varied structural trends from the involved surfactant and lipidic components. Modulations in their structural properties can be attributed to the surfactant's chemical specificity in relation to POPC, such as the headgroup hydration and the hydrophobic chain tail mismatch. Taken together, our results illustrate how specific chemical complexities of surfactant-lipid interactions can alter the morphologies of mixed mesophases and thereby alter the kinetic pathways by which surfactants dissolve lipid mesophases in bulk aqueous solutions.

Rapid and Sequential Dual Oxime Ligation Enables De Novo Formation of Functional Synthetic Membranes from Water-Soluble Precursors.

Cell membranes define the boundaries of life and primarily consist of phospholipids. Living organisms assemble phospholipids by enzymatically coupling two hydrophobic tails to a soluble polar head group. Previous studies have taken advantage of micellar assembly to couple single-chain precursors, forming non-canonical phospholipids. However, biomimetic nonenzymatic coupling of two alkyl tails to a polar head-group remains challenging, likely due to the sluggish reaction kinetics of the initial coupling step. Here we demonstrate rapid de novo formation of biomimetic liposomes in water using dual oxime bond formation between two alkyl chains and a phosphocholine head group. Membranes can be generated from non-amphiphilic, water-soluble precursors at physiological conditions using micromolar concentrations of precursors. We demonstrate that functional membrane proteins can be reconstituted into synthetic oxime liposomes from bacterial extracts in the absence of detergent-like molecules.

Controlling Protein Enrichment in Lipid Sponge Phase Droplets using SNAP-Tag Bioconjugation.

All cells use organized lipid compartments to facilitate specific biological functions. Membrane-bound organelles create defined spatial environments that favor unique chemical reactions while isolating incompatible biological processes. Despite the fundamental role of cellular organelles, there is a scarcity of methods for preparing functional artificial lipid-based compartments. Here, we demonstrate a robust bioconjugation system for sequestering proteins into zwitterionic lipid sponge phase droplets. Incorporation of benzylguanine (BG)-modified phospholipids that form stable covalent linkages with an O6 -methylguanine DNA methyltransferase (SNAP-tag) fusion protein enables programmable control of protein capture. We show that this methodology can be used to anchor hydrophilic proteins at the lipid-aqueous interface, concentrating them within an accessible but protected chemical environment. SNAP-tag technology enables the integration of proteins that regulate complex biological functions in lipid sponge phase droplets, and should facilitate the development of advanced lipid-based artificial organelles.

Non-Polar Lipids as Regulators of Membrane Properties in Archaeal Lipid Bilayer Mimics.

The modification of archaeal lipid bilayer properties by the insertion of apolar molecules in the lipid bilayer midplane has been proposed to support cell membrane adaptation to extreme environmental conditions of temperature and hydrostatic pressure. In this work, we characterize the insertion effects of the apolar polyisoprenoid squalane on the permeability and fluidity of archaeal model membrane bilayers, composed of lipid analogues. We have monitored large molecule and proton permeability and Laurdan generalized polarization from lipid vesicles as a function of temperature and hydrostatic pressure. Even at low concentration, squalane (1 mol%) is able to enhance solute permeation by increasing membrane fluidity, but at the same time, to decrease proton permeability of the lipid bilayer. The squalane physicochemical impact on membrane properties are congruent with a possible role of apolar intercalants on the adaptation of Archaea to extreme conditions. In addition, such intercalant might be used to cheaply create or modify chemically resistant liposomes (archeaosomes) for drug delivery.

Characterisation of a synthetic Archeal membrane reveals a possible new adaptation route to extreme conditions.

It has been proposed that adaptation to high temperature involved the synthesis of monolayer-forming ether phospholipids. Recently, a novel membrane architecture was proposed to explain the membrane stability in polyextremophiles unable to synthesize such lipids, in which apolar polyisoprenoids populate the bilayer midplane and modify its physico-chemistry, extending its stability domain. Here, we have studied the effect of the apolar polyisoprenoid squalane on a model membrane analogue using neutron diffraction, SAXS and fluorescence spectroscopy. We show that squalane resides inside the bilayer midplane, extends its stability domain, reduces its permeability to protons but increases that of water, and induces a negative curvature in the membrane, allowing the transition to novel non-lamellar phases. This membrane architecture can be transposed to early membranes and could help explain their emergence and temperature tolerance if life originated near hydrothermal vents. Transposed to the archaeal bilayer, this membrane architecture could explain the tolerance to high temperature in hyperthermophiles which grow at temperatures over 100 °C while having a membrane bilayer. The induction of a negative curvature to the membrane could also facilitate crucial cell functions that require high bending membranes.

Apolar Polyisoprenoids Located in the Midplane of the Bilayer Regulate the Response of an Archaeal-Like Membrane to High Temperature and Pressure.

Archaea are known to inhabit some of the most extreme environments on Earth. The ability of archaea possessing membrane bilayers to adapt to high temperature (>85°C) and high pressure (>1,000 bar) environments is proposed to be due to the presence of apolar polyisoprenoids at the midplane of the bilayer. In this work, we study the response of this novel membrane architecture to both high temperature and high hydrostatic pressure using neutron diffraction. A mixture of two diether, phytanyl chain lipids (DoPhPC and DoPhPE) and squalane was used to model this novel architecture. Diffraction data indicate that at high temperatures a stable coexistence of fluid lamellar phases exists within the membrane and that stable coexistence of these phases is also possible at high pressure. Increasing the amount of squalane in the membrane regulates the phase separation with respect to both temperature and pressure, and also leads to an increase in the lamellar repeat spacing. The ability of squalane to regulate the ultrastructure of an archaea-like membrane at high pressure and temperature supports the hypothesis that archaea can use apolar lipids as an adaptive mechanism to extreme conditions.

Lipid Phase Separation Induced by the Apolar Polyisoprenoid Squalane Demonstrates Its Role in Membrane Domain Formation in Archaeal Membranes.

Archaea synthesize methyl-branched, ether phospholipids, which confer the archaeal membrane exceptional physicochemical properties. A novel membrane organization was proposed recently to explain the thermal and high pressure tolerance of the polyextremophilic archaeon Thermococcus barophilus. According to this theoretical model, apolar molecules could populate the midplane of the bilayer and could alter the physicochemical properties of the membrane, among which is the possibility to form membrane domains. We tested this hypothesis using neutron diffraction on a model archaeal membrane composed of two archaeal diether lipids with phosphocholine and phosphoethanolamine headgroups in the presence of the apolar polyisoprenoid squalane. We show that squalane is inserted in the midplane at a maximal concentration between 5 and 10 mol % and that squalane can modify the lateral organization of the membrane and induces the coexistence of separate phases. The lateral reorganization is temperature- and squalane concentration-dependent and could be due to the release of lipid chain frustration and the induction of a negative curvature in the lipids.

Structural Characterization of an Archaeal Lipid Bilayer as a Function of Hydration and Temperature.

Archaea, the most extremophilic domain of life, contain ether and branched lipids which provide extraordinary bilayer properties. We determined the structural characteristics of diether archaeal-like phospholipids as functions of hydration and temperature by neutron diffraction. Hydration and temperature are both crucial parameters for the self-assembly and physicochemical properties of lipid bilayers. In this study, we detected non-lamellar phases of archaeal-like lipids at low hydration levels, and lamellar phases at levels of 90% relative humidity or more exclusively. Moreover, at 90% relative humidity, a phase transition between two lamellar phases was discernible. At full hydration, lamellar phases were present up to 70ᵒC and no phase transition was observed within the temperature range studied (from 25 °C to 70 °C). In addition, we determined the neutron scattering length density and the bilayer's structural parameters from different hydration and temperature conditions. At the highest levels of hydration, the system exhibited rearrangements on its corresponding hydrophobic region. Furthermore, the water uptake of the lipids examined was remarkably high. We discuss the effect of ether linkages and branched lipids on the exceptional characteristics of archaeal phospholipids.

Induction of non-lamellar phases in archaeal lipids at high temperature and high hydrostatic pressure by apolar polyisoprenoids.

It is now well established that cell membranes are much more than a barrier that separate the cytoplasm from the outside world. Regarding membrane's lipids and their self-assembling, the system is highly complex, for example, the cell membrane needs to adopt different curvatures to be functional. This is possible thanks to the presence of non-lamellar-forming lipids, which tend to curve the membrane. Here, we present the effect of squalane, an apolar isoprenoid molecule, on an archaea-like lipid membrane. The presence of this molecule provokes negative membrane curvature and forces lipids to self-assemble under inverted cubic and inverted hexagonal phases. Such non-lamellar phases are highly stable under a broad range of external extreme conditions, e.g. temperatures and high hydrostatic pressures, confirming that such apolar lipids could be included in the architecture of membranes arising from cells living under extreme environments.

The first study on the impact of osmolytes in whole cells of high temperature-adapted microorganisms.

The hyperthermophilic piezophile, Thermococcus barophilus displays a strong stress response characterized by the accumulation of the organic osmolyte, mannosylglycerate during growth under sub-optimal pressure conditions (0.1 MPa). Taking advantage of this known effect, the impact of osmolytes in piezophiles in an otherwise identical cellular context was investigated, by comparing T. barophilus cells grown under low or optimal pressures (40 MPa). Using neutron scattering techniques, we studied the molecular dynamics of live cells of T. barophilus at different pressures and temperatures. We show that in the presence of osmolytes, cells present a higher diffusion coefficient of hydration water and an increase of bulk water motions at a high temperature. In the absence of osmolytes, the T. barophilus cellular dynamics is more responsive to high temperature and high hydrostatic pressure. These results therefore give clear evidence for a protecting effect of osmolytes on proteins.

In Search for the Membrane Regulators of Archaea.

Membrane regulators such as sterols and hopanoids play a major role in the physiological and physicochemical adaptation of the different plasmic membranes in Eukarya and Bacteria. They are key to the functionalization and the spatialization of the membrane, and therefore indispensable for the cell cycle. No archaeon has been found to be able to synthesize sterols or hopanoids to date. They also lack homologs of the genes responsible for the synthesis of these membrane regulators. Due to their divergent membrane lipid composition, the question whether archaea require membrane regulators, and if so, what is their nature, remains open. In this review, we review evidence for the existence of membrane regulators in Archaea, and propose tentative location and biological functions. It is likely that no membrane regulator is shared by all archaea, but that they may use different polyterpenes, such as carotenoids, polyprenols, quinones and apolar polyisoprenoids, in response to specific stressors or physiological needs.