Modern proteins are remarkable polymers built from a 20-amino-acid alphabet, shaped by billions of years of evolution. Yet in Earth's prebiotic era, several amino acids - particularly the canonical basic residues lysine, arginine, and histidine - were likely scarce, unlike the more readily available acidic amino acids. Moreover, protein-length polymers were inaccessible before ribosomal synthesis emerged, and peptides were probably short, statistical, and non-templated. How the earliest proteins and enzymes emerged under these constraints remains a central question in origins-of-life research. Here, we synthesize random peptide libraries that span a broad electrostatic spectrum and systematically interrogate their properties. The data indicate that a prebiotically plausible acidic alphabet stands out in its propensity for secondary structure and higher-order soluble assembly via formation of β-sheets. These assemblies arise from highly heterogeneous sequences, plausibly reflecting the statistical diversity of early Earth peptides, and differ from amyloid structures in both solubility and morphology. Our results further show that the acidic random peptides have inherent capacity to bind certain metal ions, implying their potential to contribute to prebiotic catalysis. Using a large language model for structural prediction, we further show that peptides composed of this acidic alphabet exhibit a strong propensity for compact conformations. Altogether, this study showcases that unevolved sequences of prebiotically-abundant amino acids can readily produce foldable self-assembling polymers, potentially providing a steppingstone toward the first proteins, prior to the onset of purifying selection.

Department of Biological Sciences University of Maryland Baltimore County Baltimore MD 21250 USA

Department of Cell Biology Faculty of Science Charles University Prague 12800 Czech Republic

Department of Chemistry Johns Hopkins University Baltimore MD 21218 USA

Department of Physical Chemistry Faculty of Science Charles University Prague 12843 Czech Republic

Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Vestec 25250 Czech Republic

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Prague 16610 Czech Republic

School of Molecular Sciences Arizona State University Tempe AZ USA

T C Jenkins Department of Biophysics Johns Hopkins University Baltimore MD 21218 USA

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Weber A. L., Miller S. L., Reasons for the occurrence of the twenty coded protein amino acids. J Mol Evol 17, 273–284 (1981). PubMed

Cleaves H. J. II, The origin of the biologically coded amino acids. Journal of Theoretical Biology 263, 490–498 (2010). PubMed

Philip G. K., Freeland S. J., Did Evolution Select a Nonrandom “Alphabet” of Amino Acids? Astrobiology 11, 235–240 (2011). PubMed

Mayer-Bacon C., Agboha N., Muscalli M., Freeland S., Evolution as a Guide to Designing xeno Amino Acid Alphabets. International Journal of Molecular Sciences 22, 2787 (2021). PubMed PMC

Brown S. M., Mayer-Bacon C., Freeland S., Xeno Amino Acids: A Look into Biochemistry as We Do Not Know It. Life 13, 2281 (2023). PubMed PMC

Miller S. L., Urey H. C., Organic Compound Synthesis on the Primitive Earth. Science 130, 245–251 (1959). PubMed

Johnson A. P., et al. , The Miller Volcanic Spark Discharge Experiment. Science 322, 404–404 (2008). PubMed

Blanco C., Bayas M., Yan F., Chen I. A., Analysis of Evolutionarily Independent Protein-RNA Complexes Yields a Criterion to Evaluate the Relevance of Prebiotic Scenarios. Current Biology 28, 526–537.e5 (2018). PubMed

Elsila J. E., et al. , Meteoritic Amino Acids: Diversity in Compositions Reflects Parent Body Histories. ACS Cent. Sci. 2, 370–379 (2016). PubMed PMC

Glavin D. P., et al. , Abundant ammonia and nitrogen-rich soluble organic matter in samples from asteroid (101955) Bennu. Nat Astron 9, 199–210 (2025). PubMed PMC

Naraoka H., et al. , Soluble organic molecules in samples of the carbonaceous asteroid (162173) Ryugu. Science 379, eabn9033 (2023). PubMed

Higgs P. G., Pudritz R. E., A Thermodynamic Basis for Prebiotic Amino Acid Synthesis and the Nature of the First Genetic Code. Astrobiology 9, 483–490 (2009). PubMed

Despotović D., et al. , Polyamines Mediate Folding of Primordial Hyperacidic Helical Proteins. Biochemistry 59, 4456–4462 (2020). PubMed PMC

Tretyachenko V., et al. , Modern and prebiotic amino acids support distinct structural profiles in proteins. Open Biology 12, 220040 (2022). PubMed PMC

Giacobelli V. G., et al. , In Vitro Evolution Reveals Noncationic Protein–RNA Interaction Mediated by Metal Ions. Mol Biol Evol 39, msac032 (2022). PubMed PMC

Tanaka J., Doi N., Takashima H., Yanagawa H., Comparative characterization of random-sequence proteins consisting of 5, 12, and 20 kinds of amino acids. Protein Science 19, 786–795 (2010). PubMed PMC

Newton M. S., Morrone D. J., Lee K.-H., Seelig B., Genetic Code Evolution Investigated through the Synthesis and Characterisation of Proteins from Reduced-Alphabet Libraries. ChemBioChem 20, 846–856 (2019). PubMed

Giacobelli V. G., et al. , Ancient amino acid sets enable stable protein folds. [Preprint] (2025). Available at: https://www.biorxiv.org/content/10.1101/2025.10.29.685319v1 [Accessed 30 October 2025]. DOI

Meierhenrich U. J., Muñoz Caro G. M., Bredehöft J. H., Jessberger E. K., Thiemann W. H.-P., Identification of diamino acids in the Murchison meteorite. Proceedings of the National Academy of Sciences 101, 9182–9186 (2004).

Makarov M., et al. , Early Selection of the Amino Acid Alphabet Was Adaptively Shaped by Biophysical Constraints of Foldability. J. Am. Chem. Soc. 145, 5320–5329 (2023). PubMed PMC

Leman L., Orgel L., Ghadiri M. R., Carbonyl Sulfide-Mediated Prebiotic Formation of Peptides. Science 306, 283–286 (2004). PubMed

Forsythe J. G., et al. , Ester-Mediated Amide Bond Formation Driven by Wet–Dry Cycles: A Possible Path to Polypeptides on the Prebiotic Earth. Angewandte Chemie International Edition 54, 9871–9875 (2015). PubMed PMC

Canavelli P., Islam S., Powner M. W., Peptide ligation by chemoselective aminonitrile coupling in water. Nature 571, 546–549 (2019). PubMed

Foden C. S., et al. , Prebiotic synthesis of cysteine peptides that catalyze peptide ligation in neutral water. Science 370, 865–869 (2020). PubMed

Zhang Y., Cremer P. S., Interactions between macromolecules and ions: the Hofmeister series. Current Opinion in Chemical Biology 10, 658–663 (2006). PubMed

Kherb J., Flores S. C., Cremer P. S., Role of Carboxylate Side Chains in the Cation Hofmeister Series. J. Phys. Chem. B 116, 7389–7397 (2012). PubMed

Greenwald J., Kwiatkowski W., Riek R., Peptide Amyloids in the Origin of Life. Journal of Molecular Biology 430, 3735–3750 (2018). PubMed

Frenkel-Pinter M., Samanta M., Ashkenasy G., Leman L. J., Prebiotic Peptides: Molecular Hubs in the Origin of Life. Chem. Rev. 120, 4707–4765 (2020). PubMed

Malkov S. N., Živković M. V., Beljanski M. V., Hall M. B., Zarić S. D., A reexamination of the propensities of amino acids towards a particular secondary structure: classification of amino acids based on their chemical structure. J Mol Model 14, 769–775 (2008). PubMed

Pearson R. G., Hard and Soft Acids and Bases. J. Am. Chem. Soc. 85, 3533–3539 (1963).

Dokmanić I., Šikić M., Tomić S., Metals in proteins: correlation between the metal-ion type, coordination number and the amino-acid residues involved in the coordination. Acta Cryst D 64, 257–263 (2008). PubMed

Lin Z., et al. , Evolutionary-scale prediction of atomic-level protein structure with a language model. Science 379, 1123–1130 (2023). PubMed

Jing X., Wu F., Luo X., Xu J., Single-sequence protein structure prediction by integrating protein language models. Proceedings of the National Academy of Sciences 121, e2308788121 (2024).

Joosten R. P., et al. , A series of PDB related databases for everyday needs. Nucleic Acids Research 39, D411–D419 (2011). PubMed PMC

Abraham M. J., et al. , GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1–2, 19–25 (2015).

Heffernan R., et al. , Single-sequence-based prediction of protein secondary structures and solvent accessibility by deep whole-sequence learning. Journal of Computational Chemistry 39, 2210–2216 (2018). PubMed

Cuff J. A., Barton G. J., Application of multiple sequence alignment profiles to improve protein secondary structure prediction. Proteins: Structure, Function, and Bioinformatics 40, 502–511 (2000).

Garnier J., Osguthorpe D. J., Robson B., Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. Journal of Molecular Biology 120, 97–120 (1978). PubMed

Cagiada M., Ovchinnikov S., Lindorff-Larsen K., Predicting absolute protein folding stability using generative models. Protein Science 34, e5233 (2025). PubMed PMC

Sanchez-Rocha A. C., Makarov M., Pravda L., Novotný M., Hlouchová K., Coenzyme-Protein Interactions since Early Life. eLife 13 (2024).

Kim Y. E., Hipp M. S., Bracher A., Hayer-Hartl M., Hartl F. U., Molecular Chaperone Functions in Protein Folding and Proteostasis. Annual Review of Biochemistry 82, 323–355 (2013).

Hendricks M. P., Sato K., Palmer L. C., Stupp S. I., Supramolecular Assembly of Peptide Amphiphiles. Acc. Chem. Res. 50, 2440–2448 (2017). PubMed PMC

Greenwald J., Friedmann M. P., Riek R., Amyloid Aggregates Arise from Amino Acid Condensations under Prebiotic Conditions. Angewandte Chemie International Edition 55, 11609–11613 (2016). PubMed

Rout S. K., Friedmann M. P., Riek R., Greenwald J., A prebiotic template-directed peptide synthesis based on amyloids. Nat Commun 9, 234 (2018). PubMed PMC

Maury C. P. J., Amyloid and the origin of life: self-replicating catalytic amyloids as prebiotic informational and protometabolic entities. Cell. Mol. Life Sci. 75, 1499–1507 (2018). PubMed PMC

Wieczorek R., Adamala K., Gasperi T., Polticelli F., Stano P., Small and Random Peptides: An Unexplored Reservoir of Potentially Functional Primitive Organocatalysts. The Case of Seryl-Histidine. Life (Basel) 7, 19 (2017). PubMed PMC

Rufo C. M., et al. , Short peptides self-assemble to produce catalytic amyloids. Nature Chem 6, 303–309 (2014). PubMed PMC

Edri R., et al. , From Polymerization-Enabled Folding and Assembly to Chemical Evolution: Key Processes for Emergence of Functional Polymers in the Origin of Life. Astrobiology (2025).

Bordin N., Sillitoe I., Lees J. G., Orengo C., Tracing Evolution Through Protein Structures: Nature Captured in a Few Thousand Folds. Front. Mol. Biosci. 8 (2021).

Udenfriend S., et al. , Fluorescamine: A Reagent for Assay of Amino Acids, Peptides, Proteins, and Primary Amines in the Picomole Range. Science 178, 871–872 (1972). PubMed

Young G., et al. , Quantitative mass imaging of single biological macromolecules. Science 360, 423–427 (2018). PubMed PMC