Vesicles and Other Compartments

The notion that compartments are basic structures of life existed in the literature long before the work of Maturana and Varela. Historically, the first models of compartments were the "coacervates",10 that are microscopic droplets of proteins and sugars formed by self-assembly, which since they can adsorb small molecules and transform them in a sort of primitive metabolism were supposed to be the precursor of cells. A second attempt to model primitive compartments was made by Fox11 based on "pro-teinoid" microspheres formed by the polymerization products of amino acids. These two systems have only historical significance, as most of the studies to-day on protocells are carried out with vesicles, which resemble biological cells more and permit a high degree of biochemical performance. In addition to vesicles, micelles and reverse micelles have been used to construct cellular models especially with regard to self-reproduction (see ref. 2).

Vesicles are spherical compartments ranging on average 50-1000 nm in radius and are formed when appropriate surfactants are dispersed in water. They are formed by a spherically closed semipermeable boundary, the bilayer membrane, which is self-assembled by the surfactant molecules orienting their hydrophobic tails toward the inner side ofthe membrane and exposing only their hydrophilic heads to the aqueous solvent (Fig. 12.3). Phospholipids are the most typical vesicle-forming compounds and vesicles formed by lipids are usually called liposomes. Not all phospholipids, however, yield lipid vesicles. For example, the nature of the headgroup can affect the self-assembly properties of the molecule by altering the "surfactant parameter", an empirical value that can be used to envision the most stable assembly pattern ofsurfactants. Bilayers are formed when the surfactant molecules take on a cylinder-like shape with the value of v/(a0-l) about 1.

Phosphatidylcholine vesicles as well as other phospholipid vesicles are very well known and biophysical studies have been carried out to determine their formation, stability, permeability, rigidity, molecular dynamics (such as molecular diffusion in the bilayer, vibrations out-of-the-plane ofthe membrane, rotational diffusion and flip-flop movements). To have a quantitative idea about vesicle samples, consider a suspension of10 mM POPC (1-palmitoyl-2-oleoyl-sn-glycero -3-phosphocholine) unilamellar vesicles having a mean radius of 50 nm. On average, a single vesicle is composed ofabout 90,000 lipid molecules, self-assembled into a spherical bilayer membrane. One milliliter of such suspension contains ca. 1014 vesicles, each enclosing an internal volume of ca. 0.0005 fL and the collective internal volume is about 36 ^l (3.6%). In contrast, the overall (internal and external) surface is about one square meter. Sub-micrometric lipid vesicles are therefore characterized by a high surface-to-volume ratio and many of their properties are common to those of colloids. The

Figure 12.3. Detail of the membrane bilayer. Amphiphilic molecules self-assemble in bilayer structures, where the hydrophilic, polar headgroup is exposed to aqueous solvent. The hydrophobic tails are kept together and away from the aqueous solvent. On the top-right, the so-called "surfactant parameter" and its pictorial representation in terms of basic geometrical elements (v is the molecular volume of the amphiphile, a0 the "effective" headgroup surface and l is the length). The main glycerophospholipids are shown at the bottom. The surfactant parameter predicts that in most cases they can form bilayers, but some factors as the presence of salts, or insaturations on the acyl chains can change their behavior. Properties of amphiphiles mixtures are difficult to predict.

Figure 12.3. Detail of the membrane bilayer. Amphiphilic molecules self-assemble in bilayer structures, where the hydrophilic, polar headgroup is exposed to aqueous solvent. The hydrophobic tails are kept together and away from the aqueous solvent. On the top-right, the so-called "surfactant parameter" and its pictorial representation in terms of basic geometrical elements (v is the molecular volume of the amphiphile, a0 the "effective" headgroup surface and l is the length). The main glycerophospholipids are shown at the bottom. The surfactant parameter predicts that in most cases they can form bilayers, but some factors as the presence of salts, or insaturations on the acyl chains can change their behavior. Properties of amphiphiles mixtures are difficult to predict.

chemical nature of the lipid bilayer (in terms ofpolarity) allows the emergence of selective permeability. Water, glycerin, tryptophan, a generic protein and sodium ions have relative permeability in the ratios of 109:106:102:1:1 for a phosphatidylcholine membrane; thus phospholipids bilayers are impermeable to large molecules and charged ions, whereas small uncharged molecules have higher permeabilities. It is clear therefore that a semi-permeable membrane establishes a selective regime for the uptake/release of molecular components, whereas polymers such as functional macromolecules, when formed inside a vesicle, are well stored and can exert their functions inside a vesicle.

Lipid vesicles, although very useful in constructing cellular models, might have little relevance to origins of life studies. In fact, the complexity of the phospholipid molecular structure makes it difficult to consider such molecules as plausible candidates for the formation of the first protocells even though in some cases the synthesis of phospholipids has been achieved under prebiotic conditions.2

In contrast, it has been established that fatty acids were prebioti-cally available4 and that fatty acids can spontaneously self-assemble into vesicles.12,13 Fatty acids have been synthesized under a variety of allegedly prebiotic conditions (see for example ref. 14) and have also been found in the Murchison meteorite.15 When compared to phospholipid vesicles, fatty acid vesicles show different properties that are strongly dependent on the length of the acyl chain and the number of double bonds present in the chain. Moreover, fatty acids self-assemble into vesicles or micelles according to the ionization degree of their head groups (Fig. 12.4), a behavior that can be rationalized on the basis of the surfactant parameter. Long-chain carboxylates aggregate as micelles when completely ionized (high pH), whereas fatty acids separate out from aqueous solution as oil droplets when completely un-ionized (low pH). At intermediate pH values (generally around 7.5-9.5), fatty acids and their conjugate base (the carboxylate form) are present in equivalent amounts and bilayers can be formed; this observation has been explained on the basis of the hydrogen-bonded dimer shown in Figure 12.4. This intermolecular interaction to form dimers brings about a drastic change in pK: in supramolecular membrane assemblies the pK of long-chain fatty acids is around 4-5 pH units above the pK of

pH

oil droplets

vesicles

Figure 12.4. Self-assembly structures of fatty acids. A) In general, fatty acids separates out from aqueous solution as "oil" at low pH values, form bilayers (vesicles) at intermediate pH values and form micelles at high pH values (the interval 7.5-9.5 is just an example of typical behavior). B) The different self-assembly patterns at various pH have been explained on the basis of the different protonation states of the molecule, i.e., protonated at low pH, fully ionized at high pH and forming a 1:1 acid:base dimer at intermediate pH near the pK. C) Chemical structure of oleic acid.

Figure 12.4. Self-assembly structures of fatty acids. A) In general, fatty acids separates out from aqueous solution as "oil" at low pH values, form bilayers (vesicles) at intermediate pH values and form micelles at high pH values (the interval 7.5-9.5 is just an example of typical behavior). B) The different self-assembly patterns at various pH have been explained on the basis of the different protonation states of the molecule, i.e., protonated at low pH, fully ionized at high pH and forming a 1:1 acid:base dimer at intermediate pH near the pK. C) Chemical structure of oleic acid.

short-chain water-soluble monomeric carboxylic acids (e.g., acetic acid).

From these considerations and from the relatively high solubility offatty acids (~ mM), it follows that fatty acids have a complex phase diagram and are in general more reactive than their phospholipid counterparts. This can be seen as a favorable property for fatty acids being the candidates for early protocells. In the past decades, an increasing number of reports have appeared in specialized journals and vesicles from fatty acid vesicles have been investigated using different techniques, focusing on their structure, morphology, stability, physicochemical properties and reactivity. The interested reader should refer to original papers or to recent reviews regarding these investigations.4,5,16

Among the different fatty acids, oleic acid (i.e., (Z)-octadec-9-enoic acid, Fig. 12.4) has received special attention as a model system to explore fatty acid vesicles. In the following section studies on the reactivity and the transformations ofoleic acid/oleate vesicles—often referred as oleate vesicles—will be examined with respect to key pathways, that could have relevance to the origin and development of protocells.

In addition to fatty acids, the alkyl and oligoprenyl phosphates, fatty alcohols and monoacylglycerols are also considered to be prebiotically relevant.5

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