A compendium of different types of abiotic chemical syntheses identifies a consensus set of 10 “prebiotic” α-amino acids. Before the emergence of biosynthetic pathways, this set is the most plausible resource for protein formation (i.e., proteogenesis) within the overall process of abiogenesis. An essential unsolved question regarding this prebiotic set is whether it defines a “foldable set”—that is, does it contain sufficient chemical information to permit cooperatively folding polypeptides? If so, what (if any) characteristic properties might such polypeptides exhibit? To investigate these questions, two “primitive” versions of an extant protein fold (the β-trefoil) were produced by top-down symmetric deconstruction, resulting in a reduced alphabet size of 12 or 13 amino acids and a percentage of prebiotic amino acids approaching 80%. These proteins show a substantial acidification of pI and require high salt concentrations for cooperative folding. The results suggest that the prebiotic amino acids do comprise a foldable set within the halophile environment.

This article is dedicated to the memory of Michael G. Rossmann. Dating back to the last universal common ancestor, P-loop NTPases and Rossmanns comprise the most ubiquitous and diverse enzyme lineages. Despite similarities in their overall architecture and phosphate binding motif, a lack of sequence identity and some fundamental structural differences currently designates them as independent emergences. We systematically searched for structure and sequence elements shared by both lineages. We detected homologous segments that span the first βαβ motif of both lineages, including the phosphate binding loop and a conserved aspartate at the tip of β2. The latter ligates the catalytic metal in P-loop NTPases, while in Rossmanns it binds the nucleotide’s ribose moiety. Tubulin, a Rossmann GTPase, demonstrates the potential of the β2-Asp to take either one of these two roles. While convergence cannot be completely ruled out, we show that both lineages likely emerged from a common βαβ segment that comprises the core of these enzyme families to this very day.

Significance: The first proteins emerged some 4 billion y ago, and understanding how they came about is a daunting challenge. Further complicating matters, the rules of protein structure and function derived from modern proteins may be irrelevant to their earliest ancestors. We report an integrated approach in which protein sequence, structure, and function are considered. We show that a simple function (phase separation) may have served as the basis for a complex function (specific double-stranded DNA binding), and that disordered polypeptides can give rise to structured, well-packed domains. Finally, we demonstrate that functional proteins may arise from short and simple sequences that include ornithine, an amino acid likely present in early proteins yet absent in modern proteins.

Peptide-RNA coacervates can result in the concentration and compartmentalization of simple biopolymers. Given their primordial relevance, peptide-RNA coacervates may have also been a key site of early protein evolution. However, the extent to which such coacervates might promote or suppress the exploration of novel peptide conformations is fundamentally unknown. To this end, we used electron paramagnetic resonance spectroscopy (EPR) to characterize the structure and dynamics of an ancient and ubiquitous nucleic acid binding element, the helix-hairpin-helix (HhH) motif, alone and in the presence of RNA, with which it forms coacervates. Double electron-electron resonance (DEER) spectroscopy applied to singly labeled peptides containing one HhH motif revealed the presence of dimers, even in the absence of RNA. Moreover, dimer formation is promoted upon RNA binding and was detectable within peptide-RNA coacervates. DEER measurements of spin-diluted, doubly labeled peptides in solution indicated transient α-helical character. The distance distributions between spin labels in the dimer and the signatures of α-helical folding are consistent with the symmetric (HhH)₂-Fold, which is generated upon duplication and fusion of a single HhH motif and traditionally associated with dsDNA binding. These results support the hypothesis that coacervates are a unique testing ground for peptide oligomerization and that phase-separating peptides could have been a resource for the construction of complex protein structures via common evolutionary processes, such as duplication and fusion.