Liam M. Longo

Recent evidence suggests that peptide-RNA coacervates may have buffered the emergence of folded domains from flexible peptides. As primitive peptides were likely composed of both L- and D-amino acids, we hypothesized that coacervates may have also supported the emergence of chiral control. To test this hypothesis, we compared the coacervation propensities of an isotactic (homochiral) peptide and a syndiotactic (alternating chirality) peptide, both with an identical sequence derived from the ancient helix-hairpin-helix (HhH) motif. Using electron paramagnetic resonance (EPR) spectroscopy and atomistic molecular dynamics (MD) simulations, we found that the syndiotactic peptide does not form stable dimers with high α-helicity in solution, unlike the isotactic peptide. However, both peptides do coacervate with RNA, albeit with distinct reentrant phase behaviors. Coacervation in each case is facilitated by oligomer formation, likely dimerization, upon RNA binding that promotes RNA cross-linking. Additionally, RNA cross-linking and coacervation of the syndiotactic peptide may involve α-helical conformations, according to atomistic MD simulations. Coarse-grained MD simulations indicate that the differences in reentrant phase behavior of isotactic and syndiotactic peptides are associated with differences in dimer flexibility and stability, which modulate the strength of peptide-peptide and peptide-RNA interactions and, consequently, the effectiveness of RNA cross-linking. These results illustrate how RNA binding and/or coacervation by early proteins could have promoted the transition of flexible, heterochiral peptides into folded, homochiral domains.

The protein-coding regions of eukaryotic genes are fragmented into exons that, like the genes within which they are situated, can be duplicated, deleted, or reorganized. Cataloging and organizing within-gene exon similarities is necessary for a systematic study of exon evolution and its consequences. To facilitate the study of exon duplications, we present Exonize, a computational tool that identifies and classifies coding exon duplications in annotated genomes. Exonize implements a graph-based framework to handle clusters of related exons resulting from repeated rounds of exon duplication. The interdependence between duplicated exons or groups of exons across transcripts is classified. By identifying duplication events between exonic and intronic regions, Exonize can detect unannotated or degenerate exons. To aid in data parsing and downstream analysis, the Python module exonize_analysis is provided. The application of Exonize to 20 eukaryote genomes identifies full-exon duplications in at least 4% of vertebrate genes, with more than 900 human genes having a full-exon duplication event.

If you are an experimentalist interested in studying protein histories as a graduate student in the Longo Lab at ELSI, please get in touch!

Primitive nucleic acids and peptides likely collaborated in early biochemistry. What forces drove their interactions and how did these forces shape the properties of primitive complexes? We investigated how two model primordial polypeptides associate with DNA. When peptides were coupled to a ferromagnetic substrate, DNA binding depended on the substrate’s magnetic moment orientation. Reversing the magnetic field nearly abolished binding despite complementary charges. Inverting the peptide chirality or just the cysteine residue reversed this effect. These results are attributed to the chiral-induced spin selectivity (CISS) effect, where molecular chirality and electron spin alter a protein’s electric polarizability. The presence of CISS in simple protein–DNA complexes suggests that it played a significant role in ancient biomolecular interactions. A major consequence of CISS is enhancement of the kinetic stability of protein–nucleic acid complexes. These findings reveal how chirality and spin influence bioassociation, offering insights into primitive biochemical evolution and shaping contemporary protein functions.

The helix-hairpin-helix (HhH) motif is an ancient and ubiquitous nucleic acid-binding element that has emerged as a model system for studying the evolution of dsDNA-binding domains from simple peptides that phase separate with RNA. We analyzed the entire putative evolutionary trajectory of the HhH motif – from a flexible peptide to a folded domain – for functional robustness to total chiral inversion. Against expectations, functional ‘ambidexterity’ was observed for both the phase separation of HhH peptides with RNA and binding of the duplicated (HhH)2-Fold to dsDNA. Moreover, dissociation kinetics, mutational analysis, and molecular dynamics simulations revealed overlap between the binding modes adopted by the natural and mirror-image proteins to natural dsDNA. The similarity of several dissociation phases upon chiral inversion may reflect the history of (HhH)2-Fold binding, with the ultimate emergence of a high-affinity binding mode, supported by a bridging metal ion, depopulating but not displacing more primitive (potentially ambidextrous) modes. These data underscore the surprising functional robustness of the HhH protein family and suggest that the veil between worlds with alternative chiral preferences may not be as impenetrable as is often assumed.

If you are an experimentalist interested in studying protein histories as a graduate student in the Longo Lab at ELSI, please get in touch!

At the heart of many nucleoside triphosphatases is a conserved phosphate-binding sequence motif. A current model of early enzyme evolution proposes that this 6-8 residue motif could have sparked the emergence of the very first nucleoside triphosphatases – a striking example of evolutionary continuity from simple beginnings, if true. To test this provocative model, seven disembodied Walker A-derived peptides were extensively computationally characterized. Although dynamic flickers of nest-like conformations were observed, significant structural similarity between the situated peptide and its disembodied counterpart was not detected. Simulations suggest that phosphate binding is non-specific, with a preference for GTP over orthophosphate. Control peptides with the same amino acid composition but different sequences and situated conformations behaved similarly to the Walker A peptides, revealing no indication that the Walker A sequence is privileged as a disembodied peptide. We conclude that the evolutionary history of the P-Loop NTPase family is unlikely to have started with a disembodied Walker A peptide in an aqueous environment. The limits of evolutionary continuity for this protein family must be reconsidered. Finally, we argue that motifs such as the Walker A motif may represent incomplete or fragmentary molecular fossils – the true nature of which have been eroded by time.