How did the first cells on Earth arise? In a minimal cell, a membrane separates proteins and RNA from the surrounding aqueous environment. Cell-like membranes spontaneously assemble from simple prebiotic surfactants called fatty acids. However, fatty acid membranes are unstable in solutions containing salts that were likely present in environments of the early Earth. We find that amino acids, the building blocks of proteins, bind to fatty acid membranes and stabilize them against salts. Moreover, enhanced stabilization persists after dilution as would occur when a dehydrated pool refills with water—a likely setting for the emergence of cells. In addition to explaining how the first membranes were stabilized, our findings answer how key components of the first cells colocalized.

The membranes of the first protocells on the early Earth were likely self-assembled from fatty acids. A major challenge in understanding how protocells could have arisen and withstood changes in their environment is that fatty acid membranes are unstable in solutions containing high concentrations of salt (such as would have been prevalent in early oceans) or divalent cations (which would have been required for RNA catalysis). To test whether the inclusion of amino acids addresses this problem, we coupled direct techniques of cryoelectron microscopy and fluorescence microscopy with techniques of NMR spectroscopy, centrifuge filtration assays, and turbidity measurements. We find that a set of unmodified, prebiotic amino acids binds to prebiotic fatty acid membranes and that a subset stabilizes membranes in the presence of salt and Mg 2+ . Furthermore, we find that final concentrations of the amino acids need not be high to cause these effects; membrane stabilization persists after dilution as would have occurred during the rehydration of dried or partially dried pools. In addition to providing a means to stabilize protocell membranes, our results address the challenge of explaining how proteins could have become colocalized with membranes. Amino acids are the building blocks of proteins, and our results are consistent with a positive feedback loop in which amino acids bound to self-assembled fatty acid membranes, resulting in membrane stabilization and leading to more binding in turn. High local concentrations of molecular building blocks at the surface of fatty acid membranes may have aided the eventual formation of proteins.

In a minimal cell, a membrane sequesters proteins and RNA components from the surrounding aqueous solution. Prebiotic membranes would have spontaneously self-assembled from fatty acids (1, 2), which are known to be generated by abiotic reactions and delivered to Earth by meteorites (3, 4). However, fatty acid membranes are unstable in solutions containing salt at concentrations >200 mM or divalent cations at low millimolar concentrations (5). This presents a significant challenge in establishing the plausibility that the first protocell membranes were composed of fatty acids, because salt would have been prevalent in early oceans (6) and pools and because Mg2+ (or Fe2+) is essential for RNA catalysis (7, 8). Although glycerol monoesters (9, 10), long-chain alcohols (11, 12), decylamine (13, 14), and citrate (15) increase the stability of fatty acid vesicles (cell-like structures that separate an interior volume from the bulk solution), the prebiotic availability of these agents in sufficient quantities is uncertain (16). These observations lead to the question: what molecules that were likely found in prebiotic pools and oceans might interact with and stabilize fatty acid membranes? Amino acids are the building blocks of proteins, and 10 amino acids are deemed prebiotic (17⇓⇓–20). These molecules are excellent candidates for stabilizing agents of fatty acid membranes.

An additional challenge in explaining the origin of protocells is accounting for the colocalization of proteins, RNA, and membranes as a single unit. A prevalent view is that these 3 structures were formed through separate and independent processes followed by a random event that led to their colocalization. We have proposed an alternate autoamplification scenario in which fatty acid vesicles bound and concentrated the building blocks of proteins and RNA, which in turn, stabilized fatty acid vesicles, leading to further binding of the building blocks (21, 22). If the resulting conformational constraints and increased local concentration of the building blocks facilitated formation of proteins and RNA, then the colocalization of these macromolecules with fatty acid membranes would be explained. We previously reported results with RNA bases and ribose that support part of this scenario (21). Here, we seek to complete the picture by investigating interactions between amino acids and fatty acid membranes to show that all of the major components of protocells could have self-assembled into a single unit.

Our focus is on molecules that are prebiotically plausible. Previous studies have shown evidence for interactions between fatty acid vesicles and nonprebiotic amino acids and peptides (23⇓⇓⇓–27). Here, we use decanoic acid as our fatty acid rather than longer-tailed versions that produce more stable membranes but are less prebiotically plausible. We choose unmodified prebiotic amino acids rather than versions altered to optimize specific reactions. We interrogate interactions between the decanoic acid membranes and the amino acids with multiple techniques: cryotransmission electron microscopy (cryo-TEM), fluorescence microscopy, NMR spectroscopy, centrifuge filtration assays, and turbidity measurements.

We find that several amino acids bind to fatty acid membranes and that a subset stabilizes fatty acid membranes in the presence of salt and Mg2+. Moreover, we find that an amino acid’s stabilization of the membrane persists after mixing and dilution to lower overall concentrations, alleviating the need for consistently high concentrations. Together, these results explain how protocells could have endured in the presence of salt and Mg2+ and provide a plausible mechanism by which the building blocks of proteins could have colocalized with early membranes.

Results

Amino Acids Bind to Decanoic Acid Vesicles. Evidence that amino acids bind to decanoic acid vesicles emerges from 2 types of experiments: diffusion NMR spectroscopy and filtration assays. Diffusion NMR exploits the fact that, when molecules in solution bind to surfaces (even transiently), the apparent rate at which they diffuse decreases. Of the 10 amino acids that are widely regarded as prebiotically plausible (17⇓⇓–20), we chose 3 characteristic structures to investigate with this technique: glycine has no side chain, leucine has a hydrophobic side chain, and serine has a hydrophilic side chain. The structures of the fatty acid and amino acids that we used appear in SI Appendix, Fig. S1, and the experiments are summarized in SI Appendix, Table S1. As a positive control, we chose a positively charged amino acid, lysine, which is not considered prebiotic (17⇓⇓–20). Lysine should bind to negatively charged decanoic acid headgroups. We attempted to include aspartic acid (to represent amino acids with acidic side chains), but it was insoluble under our experimental conditions. On their own in solution, amino acids move freely with single fast diffusion coefficients as shown in Fig. 1A and Table 1. When the amino acids are in the presence of decanoic acid vesicles, separate fast and slow sets of coefficients appear, which arise from the biexponential curves in Fig. 1B. The reason that the fast coefficients in Fig. 1B (with decanoic acid) are slightly lower than in Fig. 1A (without decanoic acid) is likely that decanoic acid solutions have a higher bulk viscosity than water (28). The slow set of coefficients is due to binding of amino acids to decanoic acid surfaces in accordance with other NMR diffusion results (29). This binding could be due to amino acids associating with the outer surface of vesicles and micelles (the structures of which appear in SI Appendix, Fig. S1), traversing the vesicle membrane, or associating with the inner surface of vesicles. Within Fig. 1B, stronger binding is reflected in a higher y intercept (the intensity) for the second slope; hence, leucine (which is hydrophobic) and lysine (which is positively charged) bind more strongly to decanoic acid vesicles than serine and glycine do. Fig. 1. Amino acids bind to fatty acid vesicles. (A) Lysine, leucine, glycine, and serine diffuse freely in solution with 1 characteristic diffusion coefficient (m2/s) indicated by a straight line on a plot of the square of the NMR magnetic field gradient strength (G2, where G is dB/dz in units of Gauss per centimeter and B is the magnetic field) vs. normalized peak intensity (ln I/I 0 ). The slope of the line yields the first coefficient in Table 1. (B) When the amino acids are in decanoic acid solutions containing micelles and vesicles, 2 slopes (and 2 diffusion coefficients) are distinguishable. Water, which is a negative control, because it is not expected to bind to vesicles, shows only 1 slope. (C) Amino acids are retained with decanoic acid vesicles in a centrifugal filtration assay (dark bars). Controls (light bars) were performed in the absence of decanoic acid; given that decanoic acid is a surfactant, controls may represent an overestimate of the binding to the filtration unit that occurs in the presence of decanoic acid. Error bars represent SEMs for independent experiments conducted 7 (Ser), 10 (Thr), 5 (Gly), 17 (Ala), 5 (Val), 7 (Leu), 5 (Ile), 3 (thiouracil), 3 (adenine), and 4 (controls) times. The P values for the differences between Ser, Thr, Gly, Ala, Val, Leu, and Ile and their respective controls without decanoic acid are 0.15, 0.65, 0.25, 0.15, 0.04, 0.10, and 0.04, respectively (Student’s 2-tailed t test). The P values for Ser, Thr, Gly, and Ala compared with leucine are 0.03, 0.02, 0.06, and 0.03, respecticely. Thiouracil (S2U) and adenine (A) were insufficiently soluble in the absence of decanoic acid to run controls. The hydrophobicity ranking is from ref. 40. (D) Leucine and the headgroups of decanoic acid molecules interact within a distance <5 Å in lyophilized samples. 13C{2H} REDOR dephasing occurs when decanoic acid is labeled near its carboxyl group (black symbols) and not when labeled at the terminal methyl group (gray symbols). Table 1. Translational diffusion coefficients for water and for amino acids in solutions with and without decanoic acid vesicles The diffusion coefficients in Table 1 tell us that the newly appearing set of (slow) coefficients is not merely due to encapsulation of amino acids within vesicles. In samples without vesicles, leucine's diffusion coefficient corresponds to a length scale of 18 µm within the NMR timescale of 0.3 s (via the 1-dimensional diffusion equation r2 = 2Dt, where r is the length, D is the diffusion coefficient, and t is the time). Limiting the length to 10 µm, the approximate size of a decanoic acid vesicle, would yield a diffusion coefficient of 1.7 × 10−10 m2/s, which is 2 orders of magnitude greater than for leucine in the presence of vesicles. Additional support that the slow diffusion coefficient is due to binding is that diffusion coefficients for highly mobile (e.g., unsaturated) phospholipids across the surface of bilayers have comparable values within uncertainty on the order of 10−12 to 10−11 m2/s (30). Using an independent test of binding based on centrifugal filtration, we find that the 3 most hydrophobic amino acids (valine, leucine, and isoleucine) are retained to a greater extent when they are in the presence of decanoic acid vesicles and micelles (Fig. 1C). Retention of the 4 least hydrophobic amino acids (serine, threonine, glycine, and alanine) is insignificant relative to controls and is significantly less than the retention of leucine. These results support our conclusion that association of amino acids with vesicles is indeed due to binding rather than a nonspecific effect, such as encapsulation within vesicles, because encapsulation would yield the same retention in all cases. To quantify the maximum signal that we would expect from nonbinding effects, we repeated the experiment with thiouracil (which differs from the nucleobase uracil by the substitution of a sulfur for an oxygen atom). We chose thiouracil, because it is negligibly retained with decanoate micelles, which suggests that its binding to vesicles should be low as well (21). The result, depletion in the filtrate of 0.8 ± 0.2%, indicates that no more than ∼1% of the thiouracil is depleted from the filtrate due to encapsulation in vesicles. If some or all of the ∼1% is due to binding, the amount encapsulated and unbound is even lower (Fig. 1C). Our positive control, using the same assay for detection, was the nucleobase adenine, which interacts strongly with micelles and vesicles (21). As expected, adenine is strongly retained (Fig. 1C). Our results in Fig. 1 B and C, namely that hydrophobic and positively charged amino acids bind most strongly to fatty acid vesicles, imply that the amino acids’ side chains contribute to the binding. We interrogated leucine’s mode of interaction with decanoic acid by testing if it interacts with hydrogen atoms close to the headgroup of decanoic acid, with hydrogens at the end of decanoic acid’s carbon chain, or both. To conduct this test, we used rotational echo double-resonance (REDOR) NMR spectroscopy, which measures the dipolar coupling between 13C and 2H nuclei. 13C{2H} REDOR is commonly used to probe protein–membrane interactions (31⇓⇓–34). We found that leucine interacts with a hydrogen near the headgroup of decanoic acid but not at the end of the tail (Fig. 1D and SI Appendix, Fig. S2). Specifically, we lyophilized solutions containing labeled leucine and decanoic acid vesicles. We measured the dephasing of 13C-labeled leucine by 2 versions of decanoic acid: one 2H labeled at the 2-carbon that adjoins the carboxyl group and one 2H labeled at the 10-carbon at the end of the carbon chain. We find significant dephasing in the former case (indicating that the moieties interact over distances <5 Å) and not in the latter.

Vesicle Stability Against Mg2+ Correlates with an Increase in Lamellarity. Two lines of direct evidence imply that an amino acid’s success in stabilizing individual ∼10-µm vesicles in the presence of Mg2+ correlates with an increase in the number of lamellae in each vesicle. The first line of evidence follows from the fluorescence micrographs in Figs. 2 A–C and 3 A and B, which show that individual vesicles are brighter in the presence of serine and glycine, suggesting that more decanoic acid membranes are present in each vesicle. As noted above, we know that the bright structures are not oil droplets, because they have lumens that encapsulate calcein, whereas oil droplets exclude calcein (SI Appendix, Fig. S3). Fig. 3. Serine increases vesicle lamellarity. (A and B) Vesicles in the decanoic acid solution were imaged by fluorescence microscopy without (A) and with (B) 10 mM serine. A and B show cropped sections of Fig. 2 A and B with linear contrast enhancement. (Scale bars, 10 µm.) (C) Amino acids were dissolved in the decanoic acid solution to yield 10 mM solutions. Turbidity was measured by absorbance 30 min later. pH was constant. The graph shows the change in turbidity relative to a control without amino acid. (D and E) Vesicle structure in decanoic acid solutions without (D) and with (E) 10 mM serine was imaged by cryo-TEM. Arrows indicate paucilamellar vesicles; the wedge indicates multilamellar vesicles. Cryo-TEM records images of vesicles that are 2 orders of magnitude smaller than fluorescence microscopy does, because vesicles >300 nm are not retained on TEM grids. (Scale bars, 100 nm.) (F) The fraction of vesicles with >3 lamellae is higher in decanoic acid solutions containing serine. Additional evidence that serine increases lamellarity comes from cryo-TEM, which interrogates submicrometer structures. Without amino acids, >80% of submicrometer vesicles are paucilamellar, with ≤3 nested vesicles (Fig. 3 D and F). Only ∼10% of vesicles have ≥4 lamellae. When vesicle solutions include serine, which stabilizes vesicles against Mg2+, the percentage of vesicles with ≥4 lamellae jumps to >30% (Fig. 3 E and F). The increase in lamellarity induced by serine persists after the addition of Mg2+ (SI Appendix, Fig. S6). An increase in lamellarity is also beneficial in the context of protocell growth and division. As Joyce and Szostak (16) have noted: “In contrast to the behavior of multilamellar vesicles, large unilamellar vesicles are fragile and tend to rupture with extensive loss of contents under shear stress. These features combine to make multilamellar vesicles…attractive as a means of simple, environmentally driven cycles of growth and division, requiring only episodic delivery of additional amphiphiles and a moderately turbulent environment.” The amino acid leucine presents an interesting counterpoint. Leucine does not protect large ∼10-µm vesicles against Mg2+ (Fig. 2). However, leucine does protect against flocculation of vesicles in the presence of NaCl. Therefore, stabilization against flocculation by NaCl does not seem to rely on an increase in the number of lamellae.

Serine, Glycine, and the Other Relatively Hydrophilic Amino Acids Increase Turbidity. Above, we showed that an increase in the number of lamellae of decanoic acid vesicles correlates with an increase in vesicle brightness when solutions contain serine (Fig. 3). To establish a correlation that can be measured with a higher-throughput technique, we also evaluated solution turbidity, specifically solution absorbance at 490 nm. As summarized in SI Appendix, Table S1, vesicle brightness (which we measured for solutions containing serine, glycine, and leucine) correlates with turbidity: the highest turbidities are observed when the 4 most hydrophilic amino acids (serine, threonine, glycine, and alanine) are mixed with the decanoic acid solution (Fig. 3C). In these turbidity experiments, the decanoic acid solution was added to solid amino acids. Mineral surfaces (36) and ionic strength (37) are known to affect vesicle formation. To test whether the high turbidity of solutions containing the more hydrophilic amino acids results from interactions of the decanoic acid with the surfaces of the solid amino acid or from altered conditions (pH or ionic strength) in the vicinity of the dissolving amino acid, we repeated our experiments by adding amino acids as concentrated solutions rather than solids. We found that amino acids added as solutions produce the same results as when amino acids are added as solids: turbidity increases, and the most hydrophilic amino acid produces the highest increase (SI Appendix, Fig. S7). Increases in solution turbidity due to amino acids can be long lived and can arise at low concentrations. For example, when decanoic acid solutions are prepared with 10 mM serine, elevated turbidity persists at least 4 d (SI Appendix, Fig. S8), and a significant increase occurs with serine concentrations as low as 1.25 mM (SI Appendix, Fig. S9). The cryo-TEM images in Fig. 3 D and E suggest that the serine-induced increase in turbidity is due to an increase in vesicle lamellarity. This interpretation is consistent with a recent theoretical analysis predicting a strong effect of lamellarity on turbidity (38). We rule out that the increased turbidity is due to oil drops forming (SI Appendix, Fig. S3) or a decrease in the minimum concentration at which decanoic acid forms vesicles (SI Appendix, Fig. S10). In addition, we do not observe a significant increase in the size of individual vesicles either by fluorescence microscopy (Fig. 2 and SI Appendix, Fig. S3) or by dynamic light scattering analysis of the apparent hydrodynamic diameter (which was 206 nm for decanoic acid vesicles without amino acid and 205 nm with 10 mM serine). We know that the mechanism by which serine (or any other amino acid that we tested) increases lamellarity and turbidity is not through a change in solution pH, because the pH values were the same before and after the addition of amino acid.