ARTICLE
Abiotic formation of glycine-alanine peptides in alkaline evaporative environments
2024 Volume 58 Issue 5 Pages 217-226
Details
2024 Volume 58 Issue 5 Pages 217-226
Proteins are composed of 20 different amino acids and are essential catalysts of many biological reactions for all extant life. Thus, understanding the origin of proteins is essential to understanding the origin of life. Glycine (Gly) is regarded as a reactive amino acid in peptide synthesis and is also important as the most abundant α-amino acid on the prebiotic Earth. Many investigations on peptide formation have revealed the importance of evaporative environments, reporting the formation of oligopeptides composed of both single amino acids (homo-oligopeptides) and multiple-type amino acids (hetero-oligopeptides). However, the extent of incorporation of amino acids of different reactivities in peptides is unclear. We performed long-period amino acid oligomerization experiments, simulating prebiotic alkaline evaporative environments. Alkaline aqueous solutions containing a 9:5 molar ratio (pH 9.1) or 1:1 molar ratio (pH 9.2) of Gly, alanine (Ala), and sodium hydroxide (NaOH) were dried at 90°C or 130°C in glass vials over durations of 1 to 120 days. The products were analyzed with liquid chromatography-tandem mass spectrometry. The longest peptides detected were 16-mers of hetero-oligopeptides and homo-oligopeptides of Gly. The longest detected homo-oligopeptide of Ala was 4-mer. The composition of the product peptides represents a substantially higher reactivity of Gly over Ala in peptide synthesis, possibly due to the limiting of nucleophilic substitution by the bulkier side chain of Ala over Gly. Higher temperature substantially promoted the rate of the oligomerization reaction but also promoted the consumption reactions of the product oligomers. Thus, when the reaction progressed, exceeding the yield maximum of the lower temperature, the yields of peptides at the lower temperature became higher than the yields at the higher temperature, showing that longer incubation times with lower reaction temperatures are favored in the synthesis of longer and more Ala-rich peptides. Thus, even if we assume the possible higher abundance of Gly over other amino acids on Hadean Earth, these results suggest that evaporative environments over geological timescales, promoted the synthesis of polypeptides that contained glycine and other amino acids such as alanine.
Proteins are essential biological catalysts for all extant life. It has been discussed that primordial proteins spontaneously formed on the prebiotic Earth, promoting chemical evolution to the origin of life through interactions with RNA (Orgel, 1986; Cech, 2009; Tagami et al., 2017). The presence of amino acids on the early Earth has been suggested through both laboratory simulation experiments and possible extraterrestrial delivery (Glavin et al., 2010; Furukawa et al., 2009, 2015; Elsila et al., 2016; Takeuchi et al., 2020; Zang et al., 2022). Glycine (Gly) and alanine (Ala) were typically two of the most common amino acids in meteorites and in the experimental products simulating early Earth, with glycine being the most common (Glavin et al., 2010, 2020; Furukawa et al., 2009, 2015; Martins et al., 2013; Takeuchi et al., 2020).
It is not clear how amino acids polymerize to form primordial proteins on the prebiotic Hadean Earth. Many geological settings, including tidal flats, deep-sea hydrothermal vents, sea-floor sediments, comet impacts, and geothermal areas on land, have been proposed (Lahav et al., 1978; Ferris et al., 1996; Huber and Wächtershäuser, 1998; Imai et al., 1999; Kawamura et al., 2005; Ohara et al., 2007; Otake et al., 2011; Sugahara and Mimura, 2015; Kitadai et al., 2017). Among the different settings previously investigated, evaporative environments are rather advantageous in amino acid oligomerization, forming longer peptides in simulating experiments (Rodriguez-Garcia et al., 2015; Sumie et al., 2023). The longest Gly peptides detected in previous prebiotic experiments are 39-mer, resulting from the effects of boric acid under evaporative conditions (Sumie et al., 2023). Evaporative environments could also promote reactions that form components of RNA, such as ribonucleotides and ribose 5-phosphate (Lohrmann and Orgel, 1968; Burcar et al., 2016; Hirakawa et al., 2022; Takabayashi et al., 2023). Previous studies on peptide synthesis in evaporative environments mostly focused on the oligomerization of Gly, and the effects of potentially available compounds in the reaction, such as sodium hydroxide, copper, clay minerals, metal oxides, and salts (Lahav et al., 1978; Schwendinger and Rode, 1989; Saetia et al., 1993; Bujdák and Rode, 2002; Leman et al., 2004; Kitadai et al., 2011; Rodriguez-Garcia et al., 2015; Campbell et al., 2019).
Proteins contain twenty kinds of different amino acids. The sequences of the different side chain functional groups in proteins provide essential characteristics in structures and as biological catalysts, including enzymatic properties. Thus, the formation of abiotic peptides composed of multiple amino acids is substantially important. Fewer research studies have investigated hetero-oligopeptide synthesis that contains multiple amino acid species compared to homo-oligopeptide synthesis (e.g., Parker et al., 2014; Rodriguez-Garcia et al., 2015; Greenwald et al., 2016). Previous wet-dry cycle experiments under alkaline conditions identified up to 20-mer glycine homo-oligopeptides with 4-mer of hetero-oligopeptides containing Gly and Ala (Rodriguez-Garcia et al., 2015). Gly would be a more reactive amino acid for oligomerization than Ala, valine, and methionine (Otake et al., 2011; Furukawa et al., 2012; Huang et al., 2017; Rodriguez-Garcia et al., 2015). Considering the various functions that different proteinic amino acid side chains play, several amino acids likely polymerized to form early peptide sequences as precursors to create some of the initial proteins on the prebiotic Earth. However, how multiple amino acids were incorporated into peptide sequences remains unclear. We designed an experiment that investigated the formation of peptides composed of two amino acids, which have different reactivities in peptide synthesis, under drying conditions at 90°C and 130°C to understand how the difference of time, temperature, and the molar ratio of starting amino acid monomers affects the compositions of product peptides.
All commercial chemicals were used without further purification. We prepared two separate solutions at 9:5 and 1:1 molar ratios of Gly:Ala for the experiments. The molar ratio of Gly:Ala of 9:5 represents the molar ratio of their saturation in water. The Gly and l-Ala were from Wako. Water was prepared by Milli-Q integral (18.2 MΩ cm–1 and <5 ppb TOC). The stock solution of the 9:5 amino acid ratio was prepared by mixing 1000 mg of Gly and 668 mg of l-Ala with 5 mL ultra-pure water and 132.5 μL of NaOH solution containing the same mass of NaOH and H2O (pH 9.1). The stock solution of 1:1 molar ratio of Gly:Ala was prepared by mixing 562.88 mg of Gly and 668 mg of l-Ala with 5 mL ultra-pure H2O, 132.5 μL of NaOH solution containing the same mass of NaOH and H2O (pH 9.2). The NaOH of 95% purity was from Wako. From the stock solution, 102.7 μL was pipetted into uncapped glass vials. The sample solution was heated for 1, 2, 3, 4, 5, 10, 20, 30, 60, or 120 days, without rehydration at 90°C or 130°C with the lid open in an electric furnace.
Product analysisSamples were analyzed with liquid chromatography tandem mass spectrometry (LC-MS/MS; Shimadzu LCMS-8040) and Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry (FTICRMS; solariX 9.4T) equipped with a Matrix Assisted Laser Desorption/Ionization (MALDI) ionization source. For LC-MS/MS analysis, samples were rehydrated with 300 μL of water, and then vortexed for 1 min. This solution was then pipetted into a glass vial with an additional 700 μL of water, and then vortexed. This was then diluted into solutions of 1/10, 1/100, and 1/1000 concentrations with water. 200 μL of sample solution was then combined with an additional 200 μL of acetonitrile and analyzed using LC-MS/MS. Aliquots (i.e., 5 μL) of sample solutions were injected into LC. A HILIC column (VT-50-2D; Shodex) was used for separation at 55°C with an eluent flow rate of 0.25 mL/min. Isocratic elution was conducted with 70% acetonitrile and 30% 13 mM formic acid. After separation, the samples were then introduced into the mass spectrometer. Detailed conditions for MS/MS analysis were shown elsewhere (Sumie et al., 2023). Only dimers (Gly-Gly, Ala-Ala, and Gly-Ala + Ala-Gly) were quantified with commercial standards. The amounts of Ala-Gly were calculated with the calibration line of Gly-Ala.
For MALDI FTICRMS, we added 30 μL of water to an experimental vial, vortexing it for 1 min. From this, 1 μL of supernatant was pipetted and analyzed by MALDI FTICRMS. 300 scans were done with a laser irradiation count of 300 times per scan. Laser focus was set to the minimum. A total of 500 scans were performed for each sample. The data file size was set to 2 MB. The m/z range was set at m/z 202.10 to 2500.
We acquired the time-dependent profiles of all kinds of Gly-Ala peptides over the periods of their maximum yields. The initial water was mostly vaporized quickly in less than 5 minutes at 130°C and in less than 70 minutes at 90°C. Most reactions progressed after water was vaporized in the vials. The products contained a diverse range of peptides with shorter peptides being higher yields at any of the conditions. Hetero-oligopeptides of up to 16-mer containing Gly and Ala units of unidentified peptide sequences were detected (Fig. 1a, b, d). Homo-oligopeptides of Gly and Ala of up to 16-mer and 4-mer were also identified, respectively (Fig. 1c, e). At 90°C, long peptides (i.e., 12–16-mers) had the highest yield at around 60 days, whereas at 130°C the highest yields of the long peptides peaked earlier at around 20 days (Fig. 2). The length of oligomers, and numbers of Ala units in their sequence differs depending on the incubation time. When we compare the time-dependent yield profiles of product peptides at 90°C and 130°C, peptide yields had their maximum at earlier days at 130°C (Fig. 2). Peptides containing more Ala units were detected in higher yields in the products of longer incubation times (Fig. 3).
Identifications of product peptides. (a) LC-MS/MS chromatograms of 12–16-mer Gly-Ala hetero-oligopeptides containing two alanine units in their sequence. (b) LC-MS/MS chromatogram of 12–16-mer Gly-Ala hetero-oligopeptides containing one Ala unit in their sequence. (c) LC-MS/MS chromatograms of 12–16-mer Gly-Ala hetero-oligopeptides containing only glycine units in their sequence. (d) MALDI-FTICRMS spectral showing 12-mer Gly-Ala hetero-oligopeptide with 1 alanine unit in the sequence. (e) LC-MS/MS chromatogram of product Ala peptides and standards.
Time-dependent peak area of hetero-oligopeptides containing one Ala unit. Gly:Ala = 9:5 ratio. (a) 90°C, 11–16-mer in 10 μL injection analyses. (b) 130°C, 11–16-mer in 10 μL injection analyses. (c) 90°C, 7–10-mer in 5 μL injection analyses. (d) 130°C, 7–10-mer in 5 μL injection analyses. (e) 90°C, 3–6-mer in 5 μL injection analyses. (f) 130°C, 3–6-mer in 5 μL injection analyses.
LC-MS/MS peak areas of product oligomers (3-mers to 16-mers) at 30 days of reaction. (a), (b) 3-mer to 10-mer in 5 μl injection analyses, (c)–(e) 11-mer to 16-mer in 10 μl injection analyses.
The higher temperature condition at 130°C clearly promoted the reaction more rapidly (Fig. 2). For example, the maximum yields of 3–6-mer appeared at 10 days at 130°C while they appeared at 30–60 days at 90°C, when Gly and Ala of 9:5 was reacted (Fig. 2). The products were limited in shorter and Gly-rich peptides at 90°C in the early stage of reaction, whereas longer and Ala-rich peptides formed even at 90°C when the reaction progressed (Fig. 2). The yields of peptides at 90°C generally exceeded the yields of peptides at 130°C, when the reaction progressed for 120 days (Fig. 4).
LC-MS/MS peak areas of product oligomers (3-mers to 16-mers) at 120 days of reaction. (a), (b) 3-mer to 10-mer in 5 μl injection analyses, (c)–(e) 11-mer to 16-mer in 10 μl injection analyses.
The products in the experiments of 9:5 Gly:Ala generally contained larger amounts of peptides, in particular Gly-rich peptides, than that of 1:1 Gly:Ala at the same temperature and the same reaction durations (Fig. 3, 4). For example, were limited in 11-mer with 1:1 Gly:Ala conditions under 130°C at 30 days, whereas oligomers up to 16-mer were detected in the experiments of 9:5 Gly:Ala under 130°C at 30 days. One exception appeared in the experiments at 130°C for 120 days, where products from 1:1 Gly:Ala and 9:5 Gly:Ala yielded comparable amounts of peptides (Fig. 4).
Hetero-oligopeptidesUnder 130°C conditions, at both 9:5 and 1:1 molar ratio of Gly:Ala, long oligomers with one Ala unit in their sequence were generally observed to have higher peak areas than glycine homo-oligopeptides (Fig. 3, 5). For example, in the experiment of 1:1 mixed Gly:Ala molar ratio at 130°C for 30 days, the peak area of 8-mers containing 1 Ala, 2 Ala, and 3 Ala units were 2.22 times, 3.09 times, and 2.2 times higher than that of Gly 8-mer, respectively (Fig. 3b). Further, from 12-mer to 16-mer, oligomers with at least 1 Ala in their sequence were formed in larger peak area than Gly homo-oligomers (Fig. 3). Although peak areas do not represent absolute amounts, these results show the abundant incorporation of Ala in long peptides.
Product amounts of dimers at 130°C for 60 days.
Successive oligomerization progressed after water was vaporized from the vials and continued gradually over the durations investigated in the present study (Fig. 2). This indicates that the continuation of the reaction is most likely due to a combination of solid-solid reactions, which occur gradually at lower temperatures and faster at higher temperatures, and due to reactions in void water between amino acid precipitates formed by dehydration reaction. The maximum yields of oligomers appeared later in the experiments at the lower temperature (i.e., 90°C), but the maximum yields were higher at the lower temperature than at the higher temperature (i.e., 130°C). This indicates that slower reactions that occur gradually at lower temperatures are more important for the oligomerization of amino acids. Part of the amino acids and product peptides were reacted to form by-products other than longer oligomers.
The present results showed that the length of oligomers is dependent on incubation time. The highest synthesis of shorter oligopeptides resulted from short incubation times, while the highest synthesis of longer oligopeptides resulted from longer incubation times, with some longer oligomers only appearing after longer incubation times. (Fig. 2a–f). This characteristic is consistent with many previous works on the thermal oligomerization of Gly (e.g., Cleaves et al., 2009; Campbell et al., 2019). In the present study, we compared the yields of peptides at different reaction temperatures over the periods of their maximum yields and found that the yields are higher at the lower temperature when the reaction progressed exceeding the yield maximum of the lower temperature.
If the reactivities of two amino acids were the same, the products from the starting materials of the two amino acids at 1:1 would contain the same amount of homo-oligopeptides, considering the probability (Fig. 6). However, more Gly-rich oligopeptides were formed in the present study (Fig. 3, 4), showing that Gly is more reactive than Ala. This is because the bulkier side chain of Ala than Gly inhibited nucleophilic substitution of the peptide synthesis. This is consistent with many previous studies that investigated peptide synthesis of Gly and Ala, separately (Bujdák and Rode, 2002; Otake et al., 2011; Furukawa et al., 2012; Rodriguez-Garcia et al., 2015; Huang et al., 2017).
Product amounts and theoretical probabilities of dimer sequences. The probabilities of peptide formation are higher in hetero-peptides than homo-peptides due to their higher abundance of possible sequences. The higher abundance of G-G than G-A and A-G in the experiment shows the substantially higher levels of oligomerization potential of Gly compared to Ala.
Even though Gly-rich oligopeptides were formed abundantly, many hetero-oligopeptides containing Ala units were formed in this experiment (Fig. 3–5). This is because of the higher formation probabilities of hetero-oligopeptides than homo-oligopeptides (Fig. 6). This further indicates that the higher reactivity of Gly was not sufficient to prevent the formation of Ala-containing peptides.
At the early stage of the reaction, higher temperature (i.e., 130°C) appears to increase the reactivity of alanine, making higher temperatures generally more favorable for the formation of hetero-oligopeptides. The yields of hetero-oligopeptides were generally higher at the lower temperature (i.e., 90°C) than the higher temperature, when the reaction progressed (Fig. 4). This indicates that the kinetically slow nucleophilic substitution to Ala was promoted by the higher temperature. Many studies in the literature show the formation of short hetero-oligopeptides (Rodriguez-Garcia et al., 2015; Bujdák and Rode, 2002). One previous study reported the formation of long hetero-oligopeptides, including 11-mer, using COS-activated continuous polymerization (Greenwald et al., 2016). Our study showed the formation of hetero-oligopeptides of up to 16-mer in length by drying an alkaline solution.
Implications for the prebiotic EarthOxygen isotope compositions of zircon formed 4.3 billion years ago suggest that granitic protocontinents were present on the Mid-Hadean Earth (Wilde et al., 2001; Valley et al., 2014; Zhong et al., 2023). Such granitic crust might have been formed around proto-arc areas which could have offered drying environments on Hadean Earth (Furukawa and Kakegawa, 2017). Lakes in arid environments typically have alkaline pH conditions, many with a pH > 9 (Fukushi and Matsumiya, 2018). Alkaline drying environments would have been achieved around coastal areas of Hadean Earth. Hadean Earth was also characterized by heavy impacts (Cohen et al., 2000). Such impacts could have formed and provided many kinds of amino acids (Glavin et al., 2010; Furukawa et al., 2009, 2015; Elsila et al., 2016; Takeuchi et al., 2020). While the exact ratio of glycine to alanine on the prebiotic Earth is unknown, these previous studies suggest that glycine would have been the most prevalent amino acid that was synthesized and provided. For example, in the Murchison meteorite, Gly is approximately two times as abundant as Ala (Glavin et al., 2010); in simulated meteorite impact products, Gly formed at about 4 to 10 times as much as Ala (Takeuchi et al., 2020). The present study investigated the formation and the following consumption of peptides in a single batch experiment. Continuous supply of amino acids to the evaporative environments, exceeding the rate of peptide degradation, could have accumulated long peptides in evaporative environments on the prebiotic Earth.
The results of the present study show that the abundances and reactivities of amino acids affect the compositions of amino acids in abiotic peptides, indicating the formation of Gly-rich hetero-oligopeptides in Hadean alkaline drying environments. Hadean acidic conditions might have also promoted this reaction, but neutral conditions would not have promoted the reaction as suggested in previous studies (Rodriguez-Garcia et al., 2015; Sumie et al., 2023). They also suggest that even from Gly-rich amino acid pools, spontaneously formed peptides contained abundant hetero-oligopeptides. This characteristic could work for the formation of primordial polypeptides with multiple side chains.
Many hypotheses have been proposed on amino acid compositions of primordial proteins, and some propose proteins with fewer amino acids than present-day proteins, that included Gly and Ala (Crick, 1968; Wong, 1975; Akanuma et al., 2002). Some previous researchers hypothesized that prebiotic peptides contributed to constructing and expanding the ability of RNA (Noller, 2004; Cech, 2009). If this selection was affected by or inherited from the compositions of prebiotic polypeptides, the selection would have been organized by the environmental abundances of amino acids and their oligomerization reactivities as discussed in this study.
The authors appreciate Hiroyuki Momma for FTICRMS analysis. Jonathan Stimmer received a MEXT scholarship. The authors would like to thank the two anonymous reviewers and the handling editor of this manuscript for their constructive comments. This work was supported by JSPS KAKENHI (Grant No. 22H00165) to Y. F.
