Title:
SHORT AND SIMPLE: CHARTING THE EMERGENCE OF THE FIRST PROTEINS

Abstract:
The first proteins are often envisioned as having short sequences comprised of simple, abiotically-derived amino acids. However, given the complexity of modern proteins, and the lack of experimentally-validated trajectories linking them to inferred primordial states, this scenario is still largely hypothetical. During my doctoral studies, I reported the first 'prebiotic protein reconstruction' attempt: a ß-trefoil protein derived from the three-fold duplication of a 32-residue sequence element and comprised predominantly of abiotic amino acids. Although this simplified protein is well-folded, particularly under halophile conditions, it is devoid of biochemical function. Furthermore, as later studies would reveal, the ß-trefoil is a recent addition to protein fold space. To further develop the field of prebiotic protein design in my postdoctoral studies, I have engineered a functional nucleic acid binding protein comprised entirely of abiotic amino acids. This protein, dubbed ProtoH2Dup, adopts the ancient (HhH)2 fold and is the product of two identical 28-residue HhH motifs joined by a 4-residue linker. ProtoH2Dup employs just 10 amino acid types, with Ornithine (currently a non-proteogenic amino acid) serving as the sole basic amino acid type. Statistical modification of Ornithine to Arginine by a simple guanidation reaction promotes both folding and tighter DNA binding. Our results indicate that Arginine may have first emerged as a post-translational modification of Ornithine. While ProtoH2Dup has specific affinity for dsDNA, truncation to a single HhH motif yields a peptide that phase-separates in the presence of RNA. We conclude that the evolution of nucleic acid binding proteins can be seeded by relatively weak, non-specific interactions. Finally, I argue that protein simplification is a powerful tool to study the earliest stages of protein evolution - even if the exact sequences of the first proteins are lost to time.