Competition between model protocells driven by an encapsulated catalyst

Abstract

The advent of Darwinian evolution required the emergence of molecular mechanisms for the heritable variation of fitness. One model for such a system involves competing protocell populations, each consisting of a replicating genetic polymer within a replicating vesicle. In this model, each genetic polymer imparts a selective advantage to its protocell by, for example, coding for a catalyst that generates a useful metabolite. Here, we report a partial model of such nascent evolutionary traits in a system that consists of fatty-acid vesicles containing a dipeptide catalyst, which catalyses the formation of a second dipeptide. The newly formed dipeptide binds to vesicle membranes, which imparts enhanced affinity for fatty acids and thus promotes vesicle growth. The catalysed dipeptide synthesis proceeds with higher efficiency in vesicles than in free solution, which further enhances fitness. Our observations suggest that, in a replicating protocell with an RNA genome, ribozyme-catalysed peptide synthesis might have been sufficient to initiate Darwinian evolution.

Figure 1. Schematic representation of adaptive changes and competition between protocell vesicles

A: Synthesis of AcPheLeuNH₂ by catalyst encapsulated in fatty acid vesicles. A1: the dipeptide Ser-His catalyzes the reaction between substrates LeuNH₂ and AcPheOEt, generating the product of the reaction, AcPheLeuNH₂. A2: product dipeptide AcPheLeuNH₂ localizes to the bilayer membrane. B: Vesicles with AcPheLeuNH₂ in the membrane grow when mixed with vesicles without dipeptide, which shrink. C: Vesicles with AcPheLeuNH₂ in the membrane grow more following micelle addition than vesicles without the dipeptide.

Figure 2. More dipeptide synthesis and less substrate hydrolysis as a result of Ser-His catalysis in the presence of fatty acid vesicles

A: Ser-His catalyzed synthesis of AcPheLeuNH₂ is faster, and results in a higher yield, in the presence of increasing concentrations of oleate vesicles. B: Ser-His catalyzed hydrolysis of the substrate AcPheOEt is progressively slower in the presence of increasing concentrations of oleate vesicles. C: yield of dipeptide AcPheLeuNH₂ vs. concentration of oleate vesicles. D: yield of hydrolyzed substrate AcPheOH vs concentration of oleate vesicles in the same reactions. All experiments: 10mM of each substrate, 5 mM Ser-His catalyst, 0.2 M Na⁺-bicine pH 8.5, 37 °C. In these experiments, both the Ser-His catalyst and the AcPheOEt and LeuNH₂ substrates were present both inside and outside of the oleate vesicles.

Figure 3. Competition between vesicles with and without the hydrophobic dipeptide AsPheLeuNH₂

A: Self-buffered oleate vesicles (i.e. no added buffer or salt) containing the indicated mol% of AcPheLeuNH₂ were mixed with 1 or 2 equivalents of empty vesicles. After 15min the surface area was measured using the FRET-based assay. Filled markers: with FRET dyes on vesicles with AcPheLeuNH₂ growth is observed; open markers: with FRET dyes on vesicles without AcPheLeuNH₂ shrinkage is observed. B: Competition for added oleate micelles in a high-salt environment (0.2M Na⁺-bicine, pH=8.5). Equal amounts of vesicles with and without AcPheLeuNH₂ were mixed, after which oleate micelles were added, and the surface area measured after 15 min. Filled markers: FRET dyes on vesicles with AcPheLeuNH₂, growth is observed. Open markers: FRET dyes on vesicles without AcPheLeuNH₂, less growth is observed than for the corresponding peptide-containing sample. This experiment could only be carried out in the presence of added buffer, otherwise the alkaline oleate micelles (with 1 equivalent of NaOH per fatty acid, vs. the ½ equivalent per fatty acid in a vesicle) quickly and excessively changed the pH of the mixture, destabilizing the pre-formed vesicles.

Figure 4

* +: vesicles in high salt buffer (0.2M Na⁺-bicine, pH 8.5), −: self-buffered vesicles (50 mol% NaOH) ** −: no salt added; A: 50 mol% NaCl (relative to oleate), B: 100 mol% NaCl, C: 100 mol% TMAC Inhibitory effect of salt and buffer on competitive growth of vesicles. Equal amounts of oleate vesicles with and without 10 mol% AcPheLeuNH₂ were mixed and growth (or shrinkage) was monitored by a FRET-based surface area assay. Black columns: vesicles containing AcPheLeuNH₂ were labeled with FRET dyes, and growth was monitored. White columns: vesicles without AcPheLeuNH₂ were labeled with FRET dyes, and shrinkage was monitored. The presence of either salt or buffer strongly inhibits both growth and shrinkage following mixing of vesicles. Error bars indicate SEM (N=5).

Figure 5. Vesicle growth and division

A: Large multilamellar vesicles with 0.2 mol% Rh-DHPE dye and 10 mol% of dipeptide AcPheLeuNH₂ in the membrane are initially spherical. B: 10 minutes after mixing with a 100 equivalents of unlabeled, empty oleic acid vesicles without the dipeptide, thread-like filamentous structures develop. The development of filamentous vesicles from initially spherical vesicles is caused by the more rapid increase of surface area relative to volume increase, which is osmotically controlled by solute permeability. To recreate this effect in the absence of additional buffer, we used sucrose, a non-ionic osmolyte to provide an osmotic constraint on vesicle volume. C: after gentle agitation, the filamentous vesicles fragment into small daughter vesicles. Scale bar, 10 um.

Figure 6

Transmembrane pH gradient generated by growth of vesicles during competitive micelle uptake. Equal amounts of vesicles with and without 10 mol% AcPheLeuNH₂ were mixed in high salt buffer (arg⁺ -bicine, pH=8.1), and 1 equivalent of arginine-oleate micelles was added to the mixture to initiate growth. Empty circles: the peptide-containing vesicles carried the pH-sensitive water-soluble dye HPTS encapsulated in the vesicle interior. Filled circles: the peptide-free vesicles contained the HPTS. In both experiments, the two vesicle populations were mixed together and incubated for 30 min. No competitive growth at this stage, since vesicles were in a high-salt arg⁺-bicine buffer. We then added 1 equivalent of oleate-arg⁺ micelles to the mixed vesicle sample, triggering growth of both sets of vesicles. We measured the change in pH inside the vesicles vs. time by monitoring the change in the fluorescence emission of the HPTS dye (see Materials and Methods). Vesicles containing AcPheLeuNH₂ developed a larger trans-membrane pH gradient as a result of greater growth.

Figure 7. Competition between populations of protocell vesicles

A: Vesicle size changes following 1:1 mixing of indicated vesicle populations. B: Vesicle size changes following competitive oleate uptake after addition of 6 equivalents of oleate micelles. Populations of vesicles contained either Ser-His (S-H), Ser-His-Gly (S-H-G), or no catalyst, as indicated. All vesicle populations were incubated separately with amino acid substrates for 48h to allow for synthesis of hydrophobic dipeptide product prior to mixing. C: Competition between vesicles containing different catalysts. Each sample contained two or three populations of vesicles, as indicated, in a 1:1 or 1:1:1 ratio. In each case, the FRET dye pair was placed in one of the populations, to measure the size change after the reaction. Vesicles containing Ser-His outcompete vesicles containing Ser-His-Gly.