Biology subjects

Giacobelli, V. G.

Publications and source records attributed to Giacobelli, V. G..

6 recordsLinked to original sources

bioRxiv2026

Protoribosomal condensate formation across cationic chemistries

Early ribonucleoprotein systems likely required mechanisms to concentrate and organize RNA. Modern ribosomes use Mg2+ and evolved Lys/Arg-rich protein extensions to stabilize their RNA backbone. Before templated synthesis, however, peptide formation likely generated heterogeneous sequence populations rather than reproducible ribosomal sequences. Here, we compare Mg2+, ribosomal peptides, and statistical peptide libraries in organizing a 136-nucleotide model of the peptidyl transferase center (sPTC). Mg2+ condenses sPTC only at a large excess of positive charge following thermal annealing, producing largely arrested libraries. Acidic conditions further promote condensation. Ribosomal peptides instead promote coacervation near charge stoichiometry and form droplets that readily fuse. Several statistical peptide libraries also coacervate with sPTC, despite comprising heterogenous mixtures rather than a single defined sequence. Increasing mean positive charge favors condensation, but Lys/Arg-containing libraries undergo liquid-liquid phase separation more readily and across broader conditions than matched libraries containing the prebiotically plausible diaminopropionic acid (Dpr) and diaminobutyric acid (Dab). Atomistic computer simulations implicate Arg as a major source of this difference, as it can form more numerous and longer-lived hydrogen bonds with RNA, while competitive partitioning experiments show preferential condensate recruitment in the order Arg > Lys > Dab > Dpr. Together, our findings show that peptide-RNA coacervation can be triggered collectively by statistical peptide ensembles, providing a plausible route to protoribosomal organization without peptide sequence-specific optimization. Cationic chemistry shapes condensate formation and material properties, suggesting that amino acid alphabet formation could have broadened the conditions supporting liquid-like protoribosomal assemblies.

biochemistry↗

bioRxiv2025

Ancient amino acid sets enable stable protein folds

Early proteins likely arose from a chemically limited set of amino acids available through prebiotic chemistry, raising a central question in molecular evolution: could such primitive compositions yield stable, functional folds? Using de novo design, we constructed three ancient protein architectures using a reduced, evolution-inspired alphabet of ten amino acids, e.g. lacking all basic and aromatic residues. The resulting structures adopted their intended topologies and showed exceptional resistance to thermal and chemical denaturation. Computational simulations further revealed that proteins built from this restricted alphabet were as mutation-resilient as those using all twenty canonical residues. Besides their evolutionary implications, our results provide a foundation for minimalist protein design and generation of simplified, robust systems in minimal cell engineering.

biochemistry↗

bioRxiv2025

Peptides at vesicle and mineral prebiotic interfaces

The origin of life likely involved a complex interplay between organic molecules and mineral surfaces, yet the molecular details of these interactions remain poorly understood. Over recent decades, considerable research has focused on the individual roles of key biomolecules - such as RNA, lipids, and proteins - in early abiogenesis. However, this reductionist view offers only a partial picture because the emergence of life likely involved networks of molecular interactions that collectively shaped early functional assemblies. In this study, we examine the ability of peptides - arguably one of the most abundant early polymers - to interact with mineral surfaces and lipid vesicles, prebiotic interfaces and compartments. Using peptide libraries constructed from either prebiotically plausible or contemporary amino acids, we demonstrate that while acidic residues drive peptide binding to mineral surfaces (such as fluorapatite, studied here), the inclusion of arginine - a basic residue that may have been accessible in specific prebiotic environments - synergistically enhances the mobilization of bioavailable phosphate from geological reservoirs. Furthermore, we observe a functional divergence in vesicle interactions: while prebiotic alphabets promote dynamic membrane behaviours such as budding, libraries with late canonical amino acids can help preserve vesicle integrity against salt-induced collapse. Our finding supports the view that interactions with peptides can elicit changes in both prebiotic minerals and vesicles, underscoring the importance of studying these systems collectively.

evolutionary biology↗

bioRxiv2024

The interplay between peptides and RNA is critical for protoribosome compartmentalization and stability

The ribosome, owing to its exceptional conservation and biological importance, harbours a remarkable molecular fossil known as the protoribosome. It surrounds the peptidyl transferase center (PTC), responsible for peptide bond formation. While previous studies have demonstrated the PTC activity in RNA alone, our investigation reveals the intricate roles of the ribosomal protein fragments (rPeptides) within the ribosomal core. This research highlights the significance of rPeptides in stability and coacervation of two distinct protoribosomal evolutionary stages. The 617nt "big" protoribosome model which associates with rPeptides specifically, exhibits a structurally defined and rigid nature, further stabilised by the peptides. In contrast, the 136nt "small" model, previously linked to peptidyltransferase activity, displays greater structural flexibility. While this construct interacts with rPeptides with lower specificity, they induce coacervation of the "small" protoribosome across a wide concentration range, which is concomitantly dependent on the RNA sequence and structure. Moreover, these conditions protect RNA from degradation. This phenomenon suggests a significant evolutionary advantage in the RNA-protein interaction at the early stages of ribosome evolution. The distinct properties of the two protoribosomal stages suggest that rPeptides initially provided compartmentalization and prevented RNA degradation, preceding the emergence of specific RNA-protein interactions crucial for the ribosomal structural integrity. 3 keypoints- The most ancient fragments of ribosomal peptides trigger coacervation of protoribosome - The protoribosome coacervation provides protection against RNA degradation - Coacervation is more profound with a smaller and more flexible model of the protoribosome

biophysics↗

bioRxiv2021

In vitro evolution reveals primordial RNA-protein interaction mediated by metal cations

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 L11 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 L11 domain uses bridging ion (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 was compensated by acidic residues plus metal ions.

biochemistry↗

bioRxiv2020

Enzyme catalysis prior to aromatic residues: reverse engineering of a dephosphoCoA kinase

It is well-known that the large diversity of protein functions and structures is derived from the broad spectrum of physicochemical properties of the 20 canonical amino acids. According to the generally accepted hypothesis, protein evolution was continuously associated with enrichment of this alphabet, increasing stability, specificity and spectrum of catalytic functions. Aromatic amino acids are considered the latest addition to genetic code. The main objective of this study was to test whether enzymatic catalysis can spare the aromatic amino acids (aromatics) by determining the effect of amino acid alphabet reduction on structure and function of dephospho-CoA kinase (DPCK). We designed two mutant variants of a putative DPCK from Aquifex aeolicus by substituting (i) Tyr, Phe and Trp or (ii) all aromatics (including His), i.e. [~]10% of the total sequence. Their structural characterization indicates that removal of aromatic amino acids may support rich secondary structure content although inevitably impairs a firm globular arrangement. 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 is however heavily uncoupled and only one of the variants is still able to perform the reaction. Here we provide support to the hypothesis that proteins in the early stages of life could support at least some enzymatic activities, despite lower efficiencies resulting from the lack of a firm hydrophobic core. Based on the presented data we hypothesize that further protein scaffolding role may be provided by ligands upon binding. SignificanceAll extant proteins rely on the standard coded amino acid alphabet. However, early proteins lacked some of these amino acids that were incorporated into the genetic code only after the evolution of their respective metabolic pathways, aromatic amino acids being among the last additions. This is intriguing because of their crucial role in hydrophobic core packing, indispensable for enzyme catalysis. We designed two aromatics-less variants of a highly conserved enzyme from the CoA synthesis pathway, capable of enzyme catalysis and showing significant ordering upon substrate binding. To our knowledge, this is the first example of enzyme catalysis in complete absence of aromatic amino acids and presents a possible mechanism of how aromatics-less enzymes could potentially support an early biosphere.

biochemistry↗