The authors address the longstanding question of how the canonical set of proteinogenic amino acids was selected during early evolution, focusing on the cationic residues. Long cationic amino acids, notably lysine and arginine, are prevalent in modern proteins but are thought to have been scarce in prebiotic environments. Shorter basic amino acids such as ornithine (Orn) and 2,4-diaminobutyric acid (Dab) are considered more plausible prebiotic building blocks. To test whether these alternative cationic residues could support protein tertiary structure, the team designed peptide sequences intended to adopt an ancient β‑barrel architecture, the double-Ψ β-barrel (DPBB).
Design efforts combined computational protein design approaches with choices of amino acid sets that reflect plausible prebiotic chemistry, explicitly including Orn and Dab in place of lysine and arginine where relevant. The designed sequences were intended to probe whether short cationic side chains can contribute to fold stability and packing characteristic of early folded proteins.
To evaluate foldability, the researchers used a multilevel experimental strategy. They performed biophysical assays to determine whether the designed peptides adopted stable tertiary structure under various solution conditions. They complemented these experiments with high-resolution structural analysis by X‑ray crystallography to directly observe secondary and tertiary arrangements. Additionally, molecular dynamics (MD) simulations were used to model and assess structural behavior and stability at atomic detail.
The combination of computational design, biophysical characterization, crystallography, and MD provides convergent evidence on whether prebiotic cationic residues can support the formation of complex folds, and whether any observed folding corresponds to known or putative ancestral architectures.
Under typical dilute aqueous conditions used for conventional biophysical experiments, all of the designed sequences in this study remained unfolded. This outcome indicates that, in isolation and at low concentration, substitutions of modern basic residues with shorter prebiotic cationic amino acids did not by themselves produce stable, soluble folded proteins in the conditions tested.
However, one variant that contained Orn as the cationic residue displayed folding when placed in highly concentrated conditions. The authors report that this concentration-dependent folding distinguishes that Orn-containing sequence from other designed variants and highlights the importance of environmental context—such as macromolecular crowding or high solute concentration—in promoting structure formation for primitive peptides.
Crystallographic analysis was directed at the Orn-containing variant that folded under concentrated conditions and at a derivative in which Orn was replaced by Dab. Both peptides were solved by X‑ray crystallography and found to adopt a fold described by the authors as the double-Ζ β-barrel (DZBB).
The DZBB fold is characterized in the report as a plausible evolutionary intermediate between the designed DPBB target and extant β-barrel folds. The structural data therefore provide direct evidence that short, prebiotic cationic side chains can be accommodated in a compact β‑barrel–like tertiary arrangement and that small sequence changes (Orn vs Dab) can yield the same overall architecture under the tested conditions.
From these results, the authors propose that prebiotic cationic amino acids such as Orn and Dab might have supported the foldability of primitive proteins prior to the incorporation of lysine and arginine into the genetic code. The data suggest a plausible pathway by which short basic residues available on the early Earth could enable formation of stable tertiary structures—particularly under concentrated or crowded environments—potentially facilitating functional maturation of early polypeptides.
By demonstrating that an ancient β‑barrel–type architecture can be formed with prebiotically plausible cationic residues, the study supports models in which the composition of the early amino acid repertoire and environmental conditions jointly influenced the emergence of folded proteins and thereby guided subsequent selection of canonical amino acids.
This work is presented as a preprint and has not been certified by peer review. The abstract and article metadata note that the findings are preliminary pending formal peer review. Specific experimental details, quantitative stability data, and full methodological descriptions are contained in the original preprint; any findings beyond what is reported there were not provided in the source. Funding sources and author affiliations are listed in the original report.
Overall, the study uses interdisciplinary methods to show that plausible prebiotic cationic amino acids can enable formation of an ancestral β‑barrel–like fold in concentrated environments, suggesting a potential role for Orn and Dab in the early evolution of protein structure.