Recent developments in Origins of Life research have focused on substantiating the narrative of an abiotic emergence of nucleic acids from organic molecules of low molecular weight, a paradigm that typically sidelines the roles of peptides. Nevertheless, the simple synthesis of amino acids, the facile nature of their activation and condensation, their ability to recognize metals and cofactors and their remarkable capacity to self-assemble make peptides (and their analogues) favourable candidates for one of the earliest functional polymers. In this mini-review, we explore the ramifications of this hypothesis. Diverse lines of research in molecular biology, bioinformatics, geochemistry, biophysics and astrobiology provide clues about the progression and early evolution of proteins, and lend credence to the idea that early peptides served many central prebiotic roles before they were encodable by a polynucleotide template, in a putative ‘peptide-polynucleotide stage’. For example, early peptides and mini-proteins could have served as catalysts, compartments and structural hubs. In sum, we shed light on the role of early peptides and small proteins before and during the nucleotide world, in which nascent life fully grasped the potential of primordial proteins, and which has left an imprint on the idiosyncratic properties of extant proteins.

The wide variety of protein structures and functions results from the diverse properties of the 20 canonical amino acids. The generally accepted hypothesis is that early protein evolution was associated with enrichment of a primordial alphabet, thereby enabling increased protein catalytic efficiencies and functional diversification. Aromatic amino acids were likely among the last additions to genetic code. The main objective of this study was to test whether enzyme catalysis can occur without the aromatic residues (aromatics) by studying the structure and function of dephospho‐CoA kinase (DPCK) following aromatic residue depletion. We designed two variants of a putative DPCK from Aquifex aeolicus by substituting (a) Tyr, Phe and Trp or (b) all aromatics (including His). Their structural characterization indicates that substituting the aromatics does not markedly alter their secondary structures but does significantly loosen their side chain packing and increase their sizes. Both variants still possess ATPase activity, although with 150–300 times lower efficiency in comparison with the wild‐type phosphotransferase activity. The transfer of the phosphate group to the dephospho‐CoA substrate becomes heavily uncoupled and only the His‐containing variant is still able to perform the phosphotransferase reaction. These data support the hypothesis that proteins in the early stages of life could support catalytic activities, albeit with low efficiencies. An observed significant contraction upon ligand binding is likely important for appropriate organization of the active site. Formation of firm hydrophobic cores, which enable the assembly of stably structured active sites, is suggested to provide a selective advantage for adding the aromatic residues.

RNA-peptide/protein interactions have been of utmost importance to life since its earliest forms, reaching even before the last universal common ancestor (LUCA). However, the ancient molecular mechanisms behind this key biological interaction remain enigmatic because extant RNA-protein interactions rely heavily on positively charged and aromatic amino acids that were absent (or heavily under-represented) in the early pre-LUCA evolutionary period. Here, an RNA-binding variant of the ribosomal uL11 C-terminal domain was selected from a ∼1010 library of partially randomized sequences, all composed of 10 prebiotically plausible canonical amino acids. The selected variant binds to the cognate RNA with a similar overall affinity although it is less structured in the unbound form than the wild-type protein domain. The variant complex association and dissociation are both slower than for the wild-type, implying different mechanistic processes involved. The profile of the wild-type and mutant complex stabilities along with MD simulations uncover qualitative differences in the interaction modes. In the absence of positively charged and aromatic residues, the mutant uL11 domain uses ion bridging (K+/Mg2+) interactions between the RNA sugar-phosphate backbone and glutamic acid residues as an alternative source of stabilization. This study presents experimental support to provide a new perspective on how early protein-RNA interactions evolved, where the lack of aromatic/basic residues may have been compensated by acidic residues plus metal ions.

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.

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