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How Did It All Begin?
The Self-Assembly of Organic Molecules and the Origin of Cellular Life (3 of 4)
by David W. Deamer
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Self-assembly processes in prebiotic organic mixtures
The first suggestion that membranes played a role in the origin of life was in J.B.S. Haldane's prescient note in The Rationalist Annual (1926). Haldane wrote that "The cell consists of numerous half-living chemical molecules suspended in water and enclosed in an oily film. When the whole sea was a vast chemical laboratory the conditions for the formation of such films must have been relatively favorable . . ." Goldacre (1958) proposed that the first membranes could have been produced by wave action disturbing films of lipid-like surfactants. The first experimental approaches to this question were undertaken by Hargreaves et al., (1977) and Oro and co-workers (1978).
What physical properties are required if a molecule is to become incorporated into a stable bilayer? All bilayer-forming molecules are amphiphiles, with a hydrophilic "head" and a hydrophobic "tail" on the same molecule. Although we tend to think of membrane lipids as being limited to phospholipids and cholesterol, in fact a surprising variety of amphiphiles take part in membrane structure. Earlier studies (Hargreaves and Deamer, 1978) showed that even single chain amphiphiles such as alkyl phosphates, alkyl sulfates and fatty acids can assemble into bilayer membranes if they contain 10 or more carbons in their hydrocarbon chains.
If amphiphilic molecules were present in the mixture of organic compounds available on the early Earth, it is not difficult to imagine that their self-assembly into molecular aggregates was a common process. Is this a plausible premise? A reasonable start is to assume that the mixture of organic compounds in carbonaceous meteorites resembles components available on the early Earth through extraterrestrial infall. Most meteorites are composed of silicon-based minerals, and a small fraction (~5%) of these stony meteorites contain up to several percent of their mass in the form of organic carbon. These are referred to as carbonaceous meteorites, and their organic compounds are actual samples of the chemical components of the early solar system. A kerogen-like insoluble polymer composed largely of covalently linked polycyclic aromatic hydrocarbons is the most abundant organic material, while a series of organic acids (including 10-20 ppm of amino acids) represents the most abundant water-soluble fraction (Figure 1). Aliphatic and aromatic hydrocarbons, ureas, ketones, alcohols, aldehydes and purines are also present.
A variety of amphiphilic molecules could also be present in the form of polar hydrocarbon derivatives. We therefore extracted samples of the Murchison carbonaceous chondrite in the standard chloroform-methanol-water system used to extract membrane lipids from tissues. Two dimensional thin layer chromatography showed that a complex mixture of oxidized aliphatic and aromatic hydrocarbons was present. When this material was allowed to interact with aqueous phases, one class of compounds with acidic properties was clearly capable of forming membrane-bounded vesicles (Figure 2). The vesicles responded osmotically to sodium chloride or sucrose additions, and could maintain gradients of a negatively charged fluorescent dye (pyranine). This provides strong evidence that a mixture of abiotic organic compounds isolated from a carbonaceous meteorite contains amphiphiles capable of forming membranes.
Using mass spectrometry and infrared spectrophotometry (FTIR), we determined that one of the components of the mixture was nonanoic acid, a nine-carbon carboxylic acid. Nonanoic acid has too short a chain to form stable bilayers, but at neutral pH and high concentration of the amphiphile, membrane structures are readily observed by light microscopy (Figure 3). From this, we assume that the meteoritic amphiphiles contain a mixture of monocarboxylic acids such as nonanoic acid, together with polar polycyclic aromatic compounds that produce the characteristic fluorescence of the vesicle structures. Because only microscopic quantities of the membrane forming components are available, we have not been able to directly analyze the membranes themselves.
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To summarize these results, amphiphilic compounds capable of membrane formation are present in carbonaceous meteorites and are able to self-assemble into bilayer membranes. The amount of such compounds in the carbonaceous meteorites is relatively small, and we do not propose that this represents an abundant source of lipid-like material on the early Earth. However, the observation that membranes can self-assemble from the amphiphilic components at least makes it more plausible that membrane-bounded structures were present at the time of life's origin.
A model protocell
The fact that amphiphilic compounds present in meteorites can self-assemble into bilayer membranes makes it plausible that lipid-like molecules were available on the early Earth and could provide the first cellular boundary structures. The next step is to develop laboratory models of simple cellular systems in which macromolecules are encapsulated by lipid bilayers.
Liposomes are self-assembled spherical lipid bilayers in the size range of bacteria, and provide a useful model system for studies relating to the origin of cellular life. Liposomes are able to capture large molecules such as enzymes and nucleic acids, but their bilayers are relatively impermeable to smaller polar and ionic solutes. In contemporary cells, growth and reproduction require the transport of nutrients across the cell membrane and employ complex protein assemblies to facilitate the transport process. Before such proteins had evolved, what mechanism was available to transport the nutrients required for cell growth?
We have found that bilayer permeability is strongly dependent on chain length. That is, shortening the chains of a given membrane lipid dramatically increases permeation rates of ionic solutes (Paula et al., 1996). We therefore prepared liposomes with lipids of intermediate chain length, using dimyristoyl phosphatidylcholine containing 14 carbon chains. These liposomes can efficiently encapsulate enzymes, yet are permeable enough to allow influx of an externally added substrate molecules.
We first chose to encapsulate an RNA polymerase called polynucleotide phosphorylase (Chakrabarti et al., 1994). This enzyme does not depend on a template to synthesize RNA. Instead, it can use nucleotide diphosphates such as ADP as both an energy source and a monomer to be incorporated into an RNA strand (Figure 5.1). In a typical experiment, the enzyme was captured in liposomes by a simulated tide pool cycle in which a mixture of the enzyme and lipids was first dried, then rehydrated in the reaction medium. Under these conditions about half of the original enzyme can be encapsulated. ADP was added to the external medium, and after an incubation period RNA synthesis was monitored both by microscopic methods and by gel electrophoresis. We found that vesicles containing the enzyme synthesized so much RNA that it could be seen inside the liposomes when stained with the fluorescent dye ethidium bromide and then observed by fluorescence microscopy. Recently we have succeeded in capturing a more complex system that includes both a catalytic polymerase and a DNA template that acts as a kind of "gene" to direct the synthesis of RNA (Monnard et al., 1999). Under these conditions a specific transcript of RNA is transcribed from the DNA template by the polymerase, and the RNA can again be visualized by fluorescence microscopy (Figure 5.2).
These results provide a useful perspective on substrate transport by primitive forms of life. In the early Earth environment, there must have been a variety of amphiphilic hydrocarbon derivatives that could self-assemble into bilayer boundary structures. However, it is not necessary to think that the structures were of the same length and permeability properties of contemporary membranes. Instead, membrane-forming amphiphiles with 12-14 carbon chains, modeled here by DMPC, would produce bilayers that are permeable enough to allow passage of ionic substrates required for polymerization of macromolecules such as RNA, yet maintain those macromolecules within a boundary. Encapsulated catalysts and information-bearing molecules would thus have access to nutrients required for growth. Furthermore, specific groupings of macromolecules would be maintained, rather than drifting apart. This would allow true selection of such groupings to occur, a process that could not as easily take place in mixtures of molecules free in solution.
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