Prebiotic Membranes Research Articles

Synthetic chemistry recreates transitional forms in prebiotic membrane evolution

Synthetic chemistry recreates transitional forms in prebiotic membrane evolution

Prebiotic membrane structures mimic the morphology of alleged early traces of life on Earth

Elucidating compositions of the first cell membranes requires experiments with molecules and chemical conditions representative of early Earth. The molecules used are described as ‘prebiotically plausible’, i.e., they could have formed through abiotic reactions before the emergence of biology. Similarly, the chemical properties of solutions in which these membranes are formed (e.g., pH, temperature, ionic strength) must represent early Earth environments. Here, using confocal and transmission electron microscopy combined with population morphometry, we show that prebiotically plausible molecules, in solutions representative of Hadean submarine alkaline hydrothermal vents, form microstructures with substantial morphological diversity. The microstructures hold the potential for use as analogues of prebiotic processes in the rock record. Additionally, many of the structures are morphologically similar to purported early microfossils, highlighting limitations of morphological interpretation in these studies. Detailed analyses of abiotic microstructures are essential for understanding the earliest life on Earth, and for interpretation of potential biosignatures from extra-terrestrial bodies.

Prebiotic Membranes and Micelles Do Not Inhibit Peptide Formation During Dehydration.

Cycles of dehydration and rehydration could have enabled formation of peptides and RNA in otherwise unfavorable conditions on the early Earth. Development of the first protocells would have hinged upon colocalization of these biopolymers with fatty acid membranes. Using atomic force microscopy, we find that a prebiotic fatty acid (decanoic acid) forms stacks of membranes after dehydration. Using LC-MS-MS (liquid chromatography-tandem mass spectrometry) with isotope internal standards, we measure the rate of formation of serine dipeptides. We find that dipeptides form during dehydration at moderate temperatures (55 °C) at least as fast in the presence of decanoic acid membranes as in the absence of membranes. Our results are consistent with the hypothesis that protocells could have formed within evaporating environments on the early Earth.

Binding of Dipeptides to Fatty Acid Membranes Explains Their Colocalization in Protocells but Does Not Select for Them Relative to Unjoined Amino Acids.

Dipeptides, which consist of two amino acids joined by a peptide bond, have been shown to have catalytic functions. This observation leads to fundamental questions relevant to the origin of life. How could peptides have become colocalized with the first protocells? Which structural features would have determined the association of amino acids and peptides with membranes? Could the association of dipeptides with protocell membranes have driven molecular evolution, favoring dipeptides over individual amino acids? Using pulsed-field gradient nuclear magnetic resonance, we find that several prebiotic amino acids and dipeptides bind to prebiotic membranes. For amino acids, the side chains and carboxylate contribute to the interaction. For dipeptides, the extent of binding is generally less than that of the constituent amino acids, implying that other mechanisms would be necessary to drive molecular evolution. Nevertheless, our results are consistent with a scheme in which the building blocks of the biological polymers colocalized with protocells prior to the emergence of RNA and proteins.

Prebiological Membranes and Their Role in the Emergence of Early Cellular Life.

Membrane compartmentalization is a fundamental feature of contemporary cellular life. Given this, it is rational to assume that at some stage in the early origins of life, membrane compartments would have potentially emerged to form a dynamic semipermeable barrier in primitive cells (protocells), protecting them from their surrounding environment. It is thought that such prebiological membranes would likely have played a crucial role in the emergence and evolution of life on the early Earth. Extant biological membranes are highly organized and complex, which is a consequence of a protracted evolutionary history. On the other hand, prebiotic membrane assemblies, which are thought to have preceded sophisticated contemporary membranes, are hypothesized to have been relatively simple and composed of single chain amphiphiles. Recent studies indicate that the evolution of prebiotic membranes potentially resulted from interactions between the membrane and its physicochemical environment. These studies have also speculated on the origin, composition, function and influence of environmental conditions on protocellular membranes as the niche parameters would have directly influenced their composition and biophysical properties. Nonetheless, the evolutionary pathways involved in the transition from prebiological membranes to contemporary membranes are largely unknown. This review critically evaluates existing research on prebiotic membranes in terms of their probable origin, composition, energetics, function and evolution. Notably, we outline new approaches that can further our understanding about how prebiotic membranes might have evolved in response to relevant physicochemical parameters that would have acted as pertinent selection pressures on the early Earth.

A Step toward Molecular Evolution of RNA: Ribose Binds to Prebiotic Fatty Acid Membranes, and Nucleosides Bind Better than Individual Bases Do.

A major challenge in understanding how biological cells arose on the early Earth is explaining how RNA and membranes originally colocalized. We propose that the building blocks of RNA (nucleobases and ribose) bound to self-assembled prebiotic membranes. We have previously demonstrated that the bases bind to membranes composed of a prebiotic fatty acid, but evidence for the binding of sugars has remained a technical challenge. Here, we used pulsed-field gradient NMR spectroscopy to demonstrate that ribose and other sugars bind to membranes of decanoic acid. Moreover, the binding of some bases is strongly enhanced when they are linked to ribose to form a nucleoside or - with the addition of phosphate - a nucleotide. This enhanced binding could have played a role in the molecular evolution leading to the production of RNA.

Surface-Assisted Self-Assembly of Fatty Acids to Cell-Like Compartments

In contemporary biological cells, lipid membrane-enclosed, spatially distinct microenvironments confine and protect biochemical reactions and other processes. On the early Earth, the formation of independent compartments is believed to have led to the encapsulation of nucleotides, possibly aiding and facilitating the emergence of life. In this context, solid surfaces have recently been investigated as potential supports for the self-assembly of prebiotic membrane containers, as significantly enhanced compartment formation was reported in the presence of solid interfaces. Particularly, the spontaneous transformation of solid-surface-adhered phospholipid reservoirs to unilamellar vesicular compartments was shown, where the deposits undergo a well-defined progression of topological changes, starting from two-dimensional molecular lipid films, via a network of interconnected lipid nanotubes to unilamellar giant compartments. However fatty acids, having only one hydrophobic tail, are structurally simpler than phospholipids, and could have been synthesized earlier than the more intricate phospholipids, and were therefore available earlier to form protocellular compartments. We show in our latest work that, starting from simple fatty acids on solid supports, the formation of protocells can occur in the same fashion as observed in earlier experiments with phospholipids. The compartments are generated from initially unstructured deposits of fatty acids on high energy surfaces, such as SiO2, where spontaneous surface wetting, film rupturing and giant vesicle formation occurs. The new findings indicate that the earlier observed surface-assisted protocell formation route is not exclusive to phospholipids, but may have been a more general pathway in the development of early life.

Intrinsic concentration cycles and high ion fluxes in self-assembled precipitate membranes.

Concentration cycles are important for bonding of basic molecular building components at the emergence of life. We demonstrate that oscillations occur intrinsically in precipitation reactions when coupled with fluid mechanics in self-assembled precipitate membranes, such as at submarine hydrothermal vents. We show that, moreover, the flow of ions across one pore in such a prebiotic membrane is larger than that across one ion channel in a modern biological cell membrane, suggesting that proto-biological processes could be sustained by osmotic flow in a less efficient prebiotic environment. Oscillations in nanoreactors at hydrothermal vents may be just right for these warm little pores to be the cradle of life.

Stable Fatty Acid Vesicles form under Low-pH Conditions and Interact with Amino Acids and Dipeptides

Understanding how biopolymers, specifically RNA and proteins, formed prebioticly is important for understanding the origin of life. These bio-molecules are composed of building blocks that would have been widespread and at very low concentrations in early oceans. Here we investigate whether fatty acid membranes, which form spontaneously in water, could have co-assembled with these building blocks, leading to their selection and concentration and to increased membrane stability. Our previous work showed that RNA bases and ribose do bind to and stabilize fatty acid vesicles [Black et al. PNAS 110, 13272 (2013)]. Similar experiments with protein building blocks have been hampered by the extreme sensitivity of vesicle formation to pH and ionic strength at pH 7.5-8.0. Our new experiments show that decanoic acid vesicle solutions produced at pH 6.8-6.9, with phosphate buffer and 0.1 M NaCl, undergo little or undetectable change in pH upon the addition of up to 10 mM of amino acids or dipeptides, and little change in structure upon addition of a further 10 mM NaCl. Using turbidity measurements to assay vesicle formation, we find that some amino acids under these conditions, including glycine and serine, increase vesicle formation, while others, including leucine, do not. Alanine causes only a small increase in turbidity, but adding the same number of alanine residues joined to form a dipeptide doubles the effect. Blocking either the amine or carboxyl end eliminates the dipeptide-induced increase, suggesting that both ends interact with the vesicle surface. Cryo-electron microscopy images indicate that in addition to altering the extent of vesicle formation, alanine-alanine increases contact between lamellae in multilamellar vesicles. Thus prebiotic membranes may have assembled with amino acids and short peptides, which in turn would have altered membrane formation and structure.

Amino Acids and Peptides Stabilize Fatty Acid Membranes against Salt-Induced Flocculation

The prebiotic formation of biopolymers (specifically DNA, RNA, and proteins) has long been a mystery and is important for understanding the origin of life on earth. These bio-molecules are composed of building blocks that would have been dispersed in early oceans. Our previous work has shown that RNA bases and ribose bind to and stabilize fatty acid vesicles [Black et al. PNAS 110, 13272 (2013)]. Our results implied that the building blocks of a biological polymer could have spontaneously associated with components of the first membranes to form stable structures. We have now shown that protein building blocks, too, stabilize fatty acid vesicles against salt-induced flocculation. Using spectrophotometry, we measured the presence of flocs (and other structures) in fatty acid solutions, with and without amino acids and over a range of temperatures. Using fluorescence microscopy, we identified the structures that caused changes in absorbance in our spectrophotometric assays. We found that the two most hydrophobic prebiotic amino acids, leucine and isoleucine, prevent salt-induced flocculation. Moreover, although alanine and glycine, which are less hydrophobic, had little effect on flocculation, dipeptides composed of these amino acids preserved vesicles in the presence of salt even at 60 degrees C. These vesicles appeared to be primarily multilamellar structures, which may promote reactions between components of biopolymers more effectively than unilamellar vesicles. Thus prebiotic membranes could have facilitated the formation of peptides by bringing amino acids together, and peptides could have increased the formation of stable membranes. Such an auto-amplifying system, combined with selection for more effective peptides, could have led to the first cells.

Prebiotic cell membranes that survive extreme environmental pressure conditions.

Attractive candidates for compartmentalizing prebiotic cells are membranes comprised of single-chain fatty acids. It is generally believed that life may have originated in the depth of the protoocean, that is, under high hydrostatic pressure conditions, but the structure and physical-chemical properties of prebiotic membranes under such conditions have not yet been explored. We report the temperature- and pressure-dependent properties of membranes composed of prebiotically highly-plausible lipids and demonstrate that prebiotic membranes could not only withstand extreme temperatures, but also serve as robust models of protocells operating in extreme pressure environments. We show that pressure not only increases the stability of vesicular systems but also limits their flexibility and permeability to solutes, while still keeping the membrane in an overall fluid-like and thus functional state.

Nucleobases and Ribose Bind to and Stabilize Aggregates of a Prebiotic Amphiphile

One scenario for the origin of life postulates that catalytic RNA can be synthesized and encapsulated in a membranous compartment to form primitive cells. Here we examine physical interactions between fatty acid aggregates, especially vesicles, and the bases and sugar composing RNA. Our goal is to employ an array of fundamental physical techniques to establish the plausibility of a scenario in which aggregates of amphiphiles preceded RNA and facilitated its synthesis by binding and concentrating the bases and sugars of which it is composed. We find that fatty acid membranes do indeed bind nucleobases and ribose more effectively than they bind several related bases and sugars. Moreover, we find that nucleobases and ribose also inhibit flocculation of fatty acids by salt, and do so more effectively than several other bases and sugars. This result is important because it addresses how prebiotic membranes could have been stabilized in early oceans with high salt concentrations. Taken together, our results yield mutually reinforcing mechanisms of adsorption, concentration and stabilization that could have driven the emergence of a population of stable vesicles enriched in the components of RNA.

Early self-reproduction, the emergence of division mechanisms in protocells

Synthetic Biology approaches are proposing model systems and providing experimental evidences that life can arise as spontaneous chemical self-assembly process where the ability to reproduce itself is an essential feature of the living system. The appearance of early cells has required an amphiphilic membrane compartment to confine molecular information against diffusion, and the ability to self-replicate the boundary layer and the genetic information. The initial spontaneous self-replication mechanisms based on thermodynamic instability would have evolved in a prebiotic and later biological catalysis. Early studies demonstrate that fatty acids spontaneously assemble into bilayer membranes, building vesicles able to grow by incorporation of free lipid molecules and divide. Early replication mechanisms may have seen inorganic molecules playing a role as the first catalysts. The emergence of a short ribozyme or short catalytic peptide may have initiated the first prebiotic membrane lipid synthesis required for vesicle growth. The evolution of early catalysts towards the simplest translation machine to deliver proteins from RNA sequences was likely to give early birth to one single enzyme controlling protocell membrane division. The cell replication process assisted by complex enzymes for lipid synthesis is the result of evolved pathways in early cells. Evolution from organic molecules to protocells and early cells, thus from chemistry to biology, may have occurred in and out of the boundary layer. Here we review recent experimental work describing membrane and vesicle division mechanisms based on chemico-physical spontaneous processes, inorganic early catalysis and enzyme based mechanisms controlling early protocell division and finally the feedback from minimal genome studies.

Polycyclic Aromatic Hydrocarbons as Plausible Prebiotic Membrane Components

Aromatic molecules delivered to the young Earth during the heavy bombardment phase in the early history of our solar system were likely to be among the most abundant and stable organic compounds available. The Aromatic World hypothesis suggests that aromatic molecules might function as container elements, energy transduction elements and templating genetic components for early life forms. To investigate the possible role of aromatic molecules as container elements, we incorporated different polycyclic aromatic hydrocarbons (PAH) in the membranes of fatty acid vesicles. The goal was to determine whether PAH could function as a stabilizing agent, similar to the role that cholesterol plays in membranes today. We studied vesicle size distribution, critical vesicle concentration and permeability of the bilayers using C6-C10 fatty acids mixed with amphiphilic PAH derivatives such as 1-hydroxypyrene, 9-anthracene carboxylic acid and 1,4 chrysene quinone. Dynamic Light Scattering (DLS) spectroscopy was used to measure the size distribution of vesicles and incorporation of PAH species was established by phase-contrast and epifluorescence microscopy. We employed conductimetric titration to determine the minimal concentration at which fatty acids could form stable vesicles in the presence of PAHs. We found that oxidized PAH derivatives can be incorporated into decanoic acid (DA) vesicle bilayers in mole ratios up to 1:10 (PAH:DA). Vesicle size distribution and critical vesicle concentration were largely unaffected by PAH incorporation, but 1-hydroxypyrene and 9-anthracene carboxylic acid lowered the permeability of fatty acid bilayers to small solutes up to 4-fold. These data represent the first indication of a cholesterol-like stabilizing effect of oxidized PAH derivatives in a simulated prebiotic membrane.

Life defined

Life defined

Neutron reflectivity study of substrate surface chemistry effects on supported phospholipid bilayer formation on [formula omitted] sapphire

Oxide-supported phospholipid bilayers (SPBs) used as biomimetic membranes are significant for a broad range of applications including improvement of biomedical devices and biosensors, and in understanding biomineralization processes and the possible role of mineral surfaces in the evolution of pre-biotic membranes. Continuous-coverage and/or stacked SPBs retain properties (e.g., fluidity) more similar to native biological membranes, which is desirable for most applications. Using neutron reflectivity, we examined the role of oxide surface charge (by varying pH and ionic strength) and of divalent Ca2+ in controlling surface coverage and potential stacking of dipalmitoylphosphatidylcholine (DPPC) bilayers on the (112¯0) face of sapphire (α-Al2O3). Nearly full bilayers were formed at low to neutral pH, when the sapphire surface is positively charged, and at low ionic strength (I=15mM NaCl). Coverage decreased at higher pH, close to the isoelectric point of sapphire, and also at high I⩾210mM, or with addition of 2mM Ca2+. The latter two effects are not additive, suggesting that Ca2+ mitigates the effect of higher I. These trends agree with previous results for phospholipid adsorption on α-Al2O3 particles determined by adsorption isotherms and on single-crystal (101¯0) sapphire by atomic force microscopy, suggesting consistency of oxide surface chemistry-dependent effects across experimental techniques.

Prebiotic Chemistry and the Origin of the RNA World

The demonstration that ribosomal peptide synthesis is a ribozyme-catalyzed reaction makes it almost certain that there was once an RNA World. The central problem for origin-of-life studies, therefore, is to understand how a protein-free RNA World became established on the primitive Earth. We first review the literature on the prebiotic synthesis of the nucleotides, the nonenzymatic synthesis and copying of polynucleotides, and the selection of ribozyme catalysts of a kind that might have facilitated polynucleotide replication. This leads to a brief outline of the Molecular Biologists' Dream, an optimistic scenario for the origin of the RNA World. In the second part of the review we point out the many unresolved problems presented by the Molecular Biologists' Dream. This in turn leads to a discussion of genetic systems simpler than RNA that might have “invented” RNA. Finally, we review studies of prebiotic membrane formation.

Self-assembled vesicles of monocarboxylic acids and alcohols: conditions for stability and for the encapsulation of biopolymers

We tested the ability of saturated n-monocarboxylic acids ranging from eight to 12 carbons in length to self-assemble into vesicles, and determined the minimal concentrations and chain lengths necessary to form stable bilayer membranes. Under defined conditions of pH and concentrations exceeding 150 mM, an unbranched monocarboxylic acid as short as eight carbons in length ( n-octanoic acid) assembled into vesicular structures. Nonanoic acid (85 mM) formed stable vesicles at pH 7.0, the p K of the acid in bilayers, and was chosen for further testing. At pH 6 and below, the vesicles were unstable and the acid was present as droplets. At pH ranges of 8 and above clear solutions of micelles formed. However, addition of small amounts of an alcohol (nonanol) markedly stabilized the bilayers, and vesicles were present at significantly lower concentrations (∼20 mM) at pH ranges up to 11. The formation of vesicles near the p K a of the acids can be explained by the formation of stable RCOO −…HOOCR hydrogen bond networks in the presence of both ionized and neutral acid functions. Similarly, the effects of alcohols at high pH suggests the formation of stable RCOO −…HOR hydrogen bond networks when neutral RCOOH groups are absent. The vesicles provided a selective permeability barrier, as indicated by osmotic activity and ionic dye capture, and could encapsulate macromolecules such as DNA and a protein. When catalase was encapsulated in vesicles of decanoic acid and decanol, the enzyme was protected from degradation by protease, and could act as a catalyst for its substrate, hydrogen peroxide, which readily diffused across the membrane. We conclude that membranous vesicles produced by mixed short chain monocarboxylic acids and alcohols are useful models for testing the limits of stabilizing hydrophobic effects in membranes and for prebiotic membrane formation.

Ion transport mechanisms and prebiotic membranes

Ion transport mechanisms and prebiotic membranes

Synthesis of phospholipids and membranes in prebiotic conditions.

IT is generally agreed that stable membranes were prerequisite to the assembly of the earliest self-replicating systems1–4. Phospholipids, which are ubiquitous in biological membranes and which self-assemble in aqueous environments into stable lipid bilayers and vesicles4, are obvious candidates for prebiotic membrane components. We report here the abiotic synthesis of various lipids, including membranogenic phospholipids.