Prebiotic amino acids bind to and stabilize prebiotic fatty acid membranes

Abstract

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 Mg²⁺ 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.

Keywords: amino acid; membrane; origin of life; prebiotic; protocell.

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/I₀). 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. . (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).

Fig. 2.

Amino acids can stabilize decanoic acid vesicles against Mg²⁺ and NaCl. (A) Vesicles ∼10 µm in diameter self-assemble in the decanoic acid solution described in Methods. (B and C) In the presence of 10 mM serine or glycine, these ∼10-µm vesicles appear brighter, consistent with multilamellar structures. Vesicle lumens are aqueous; they can be labeled with calcein, a soluble dye (SI Appendix, Fig. S3). (D) In contrast, in the presence of 10 mM leucine, ∼10-µm vesicles are indistinguishable from those in A. (E–H) When the decanoic acid solutions include 10 mM Mg²⁺, bright ∼10-µm vesicles are retained if the solutions also contain serine or glycine. In contrast, in solutions without amino acid or in the presence of leucine, micrometer-scale vesicles are replaced by punctate structures, some of which are smaller vesicles. (I–L) When 300 mM NaCl is added at room temperature to 80 mM decanoic acid at pH 7.65, vesicles flocculate. After heating the solution to 60 °C to disaggregate the flocs and then cooling to 30 °C, individual ∼5-µm vesicles reform in solutions containing 10 mM serine, glycine, or leucine. In contrast, in solutions without amino acid, flocs reform. All panels are fluorescence micrographs. (Scale bars, 10 µm.)

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.

Fig. 4.

Amino acids increase turbidity of vesicle solutions in 2 scenarios that recapitulate pools undergoing cycles of drying and wetting. (A) Drying and rehydration of the decanoic acid solution containing amino acids result in elevated turbidity (absorbance at 490 nm) relative to a solution without amino acids. Error bars show SEM for 4 experiments with glycine, alanine, and serine and show average error for 2 leucine experiments. (B) Delayed mixing of decanoic acid solutions and amino acid results in higher turbidity, even when final concentrations are constant. The result holds equally well when the amino acid is a solid and when it is in solution. In “solid” samples, the decanoic acid solution was added to a test tube containing solid amino acid such that the final amino acid concentration was 10 mM. In “solution” samples, 10 µL of 1 M amino acid in 50 mM sodium phosphate at pH 6.83 (or the buffer alone) was placed on top of 990 µL of the decanoic acid solution. For both types of samples, mixing by vortexing occurred either immediately or after a delay of 30 s. Error bars show average error for duplicate samples.