Whereas modern proteins rely on a quasi-universal repertoire of 20 canonical amino acids (AAs), numerous lines of evidence suggest that ancient proteins relied on a limited alphabet of 10 "early" AAs and that the 10 "late" AAs were products of biosynthetic pathways. However, many nonproteinogenic AAs were also prebiotically available, which begs two fundamental questions: Why do we have the current modern amino acid alphabet and would proteins be able to fold into globular structures as well if different amino acids comprised the genetic code? Here, we experimentally evaluate the solubility and secondary structure propensities of several prebiotically relevant amino acids in the context of synthetic combinatorial 25-mer peptide libraries. The most prebiotically abundant linear aliphatic and basic residues were incorporated along with or in place of other early amino acids to explore these alternative sequence spaces. The results show that foldability was likely a critical factor in the selection of the canonical alphabet. Unbranched aliphatic amino acids were purged from the proteinogenic alphabet despite their high prebiotic abundance because they generate polypeptides that are oversolubilized and have low packing efficiency. Surprisingly, we find that the inclusion of a short-chain basic amino acid also decreases polypeptides' secondary structure potential, for which we suggest a biophysical model. Our results support the view that, despite lacking basic residues, the early canonical alphabet was remarkably adaptive at supporting protein folding and explain why basic residues were only incorporated at a later stage of protein evolution.

Department of Cell Biology Faculty of Science Charles University BIOCEV Prague 12843 Czech Republic

Department of Chemistry Johns Hopkins University Baltimore Maryland 21218 United States

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

Earth Life Science Institute Tokyo Institute of Technology Tokyo 1528550 Japan

Graduate School of Media and Governance Keio University Fujisawa 2520882 Japan

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

T C Jenkins Department of Biophysics Johns Hopkins University Baltimore Maryland 21218 United States

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The interplay between peptides and RNA is critical for protoribosome compartmentalization and stability

Phosphoric acid salts of amino acids as a source of oligopeptides on the early Earth

Peptides En Route from Prebiotic to Biotic Catalysis