Biology subjects

Hlouchova, K.

Publications and source records attributed to Hlouchova, K..

14 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↗

bioRxiv2026

Learning the structural diversity in random protein sequence space

The universe of possible protein sequences is astronomically large, yet our understanding of the sequence-structure relationship is confined to the infinitesimal fraction used currently by life. Determining whether "foldable" architectures are rare singularities or accessible solutions is critical for understanding protein evolution and designing novel proteins. Here, we map the structural landscape of random sequence space by screening one million synthetic proteins using a high-throughput in vivo FRET biosensor. We reveal that this space is structurally heterogeneous, populated not only by disordered chains and stress-inducing aggregates but also by "benign" compact structures that resemble globular proteins and evade cellular chaperone responses. By training machine learning models on these phenotypes, we show that structural potential is learnable and generalizes to natural proteomes. These findings demonstrate that biology-like folds are accessible from random sequences with surprising frequency, providing data required to expand generative protein design beyond evolutionary priors.

synthetic biology↗

bioRxiv2026

Xeno amino acid alphabets form peptides with familiar secondary structure

Summary paragraphThe central dogma of molecular biology describes how genetic information stored in nucleic acids guides the formation of structured proteins from a single molecular alphabet of twenty amino acids (C20)1,2. The extent to which C20 is uniquely capable of forming structural polymers remains a fundamental open question. Here we demonstrate that peptides built from other, "xeno" amino acid alphabets can adopt protein-like secondary structural motifs. Having designed two different xeno alphabets, with them we constructed both combinatorial random sequence libraries and specific, designed 25-mer sequences. We report sequence-dependent structural motifs that result according to circular dichroism, infrared spectroscopy and nuclear magnetic resonance interpreted by molecular dynamics simulations, supported by extensive quantum mechanical calculations. This evidence, including a solution-phase NMR-resolved helical motif, demonstrates that the potential for amino acids to form structure bearing sequences is not unique to lifes alphabet, and thereby reveals a previously unexplored sequence-structure space. Results inform the search for extraterrestrial life, lay foundations for incorporating novel synthetic functional groups and heteroatoms into structure-bearing alphabets, and provide the first truly independent data with which to test and improve all that has been learned about protein folding from the study of lifes 20 side chains.

biochemistry↗

bioRxiv2025

Prebiotically Plausible Peptides can Self-assemble into β-rich Nanostructures

Peptides can self-assemble into diverse morphologies in a programmable manner and hence are privileged building blocks used widely in nanotechnology. Most reported peptide nanostructures consist of one (or a few) defined sequence(s), as self-similarity is presumed to be essential for promoting assembly. While oligomerisation is seen as an important feature of the earliest functional polymers during the origin of life, prebiotic peptides were likely short, statistical, and non-templated - traits that seem incommensurate with robust self-assembly. Here we show that random 25-mer peptides can efficiently and spontaneously form highly thermostable, soluble assemblies rich with beta-sheets. Notably, these nanostructures only emerge when random peptides are constructed with an early alphabet, consisting of the 10 canonical amino acids that were also prebiotically abundant - but not other alphabets tested. Hence, our findings show that peptide self-assembly does not require purity, and in fact compositional complexity is adaptive for preventing formation of insoluble structures. Altogether, this study showcases that unevolved sequences of prebiotically-abundant amino acids can readily produce foldable self-assembling polymers, thereby providing a potential steppingstone toward the first proteins, prior to the onset of purifying selection.

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↗

bioRxiv2024

High-throughput selection of human de novo-emerged sORFs with high folding potential

De novo genes emerge from previously non-coding stretches of the genome. Their en-coded de novo proteins are generally expected to be similar to random sequences and, accordingly, with no stable tertiary fold and high predicted disorder. However, structural properties of de novo proteins and whether they differ during the stages of emergence and fixation have not been studied in depth and rely heavily on predictions. Here we generated a library of short human putative de novo proteins of varying lengths and ages and sorted the candidates according to their structural compactness and disorder propensity. Using Forster resonance energy transfer (FRET) combined with Fluorescence-activated cell sorting (FACS) we were able to screen the library for most compact protein structures, as well as most elongated and flexible structures. Compact de novo proteins are on average slightly shorter and contain lower predicted disorder than less compact ones. The predicted structures for most and least compact de novo proteins correspond to expectations in that they contain more secondary structure content or higher disorder content, respectively. Our experiments indicate that older de novo proteins have higher compactness and structural propensity compared to young ones. We discuss possible evolutionary scenarios and their implications underlying the age-dependencies of compactness and structural content of putative de novo proteins.

evolutionary biology↗

bioRxiv2023

Coenzyme-Protein Interactions since Early Life

Recent findings in protein evolution and peptide prebiotic plausibility have been setting the stage for reconsidering the role of peptides in the early stages of lifes origin. Ancient protein families have been found to share common themes and proteins reduced in composition to prebiotically plausible amino acids have been reported capable of structure formation and key functions, such as binding to RNA. While this may suggest peptide relevance in early life, their functional repertoire when composed of a limited number of early residues (missing some of the most sophisticated functional groups of todays alphabet) has been debated. Cofactors enrich the functional scope of about half of extant enzymes but whether they could also bind to peptides lacking the evolutionary late amino acids remains speculative. The aim of this study was to resolve the early peptide propensity to bind organic cofactors by analysis of protein-coenzyme interactions across the Protein Data Bank (PDB). We find that the prebiotically plausible amino acids are more abundant in the binding sites of the most ancient coenzymes and that such interactions rely more frequently on the involvement of the protein backbone atoms and metal ion cofactors. Moreover, we have identified a few select examples in todays enzymes where coenzyme binding is supported solely by prebiotically available amino acids. These results imply the plausibility of a coenzyme-peptide functional collaboration preceding the establishment of the Central Dogma and full protein alphabet evolution.

bioinformatics↗

bioRxiv2022

Early selection of the amino acid alphabet was adaptively shaped by biophysical constraints of foldability

Whereas modern proteins rely on a quasi-universal repertoire of 20 canonical amino acids (AAs), numerous lines of evidence suggest that ancient proteins relied on a limited alphabet of 10 early AAs, and that the 10 late AAs were products of biosynthetic pathways. However, many non-proteinogenic AAs were also prebiotically available, which begs two fundamental questions: Why do we have the current modern amino acid alphabet, and Would proteins be able to fold into globular structures as well if different amino acids comprised the genetic code? Here, we experimentally evaluated the solubility and secondary structure propensities of several prebiotically relevant amino acids in the context of synthetic combinatorial 25-mer peptide libraries. The most prebiotically abundant linear aliphatic and basic residues were incorporated along with or in place of other early amino acids to explore these alternative sequence spaces. We show that foldability was a critical factor in the selection of the canonical alphabet. Unbranched aliphatic and short-chain basic amino acids were purged from the proteinogenic alphabet despite their high prebiotic abundance because they generate polypeptides that are over-solubilized and have low packing efficiency. Surprisingly, we find that the inclusion of a short-chain basic amino acid also decreases polypeptides secondary structure potential. Our results support the view that despite lacking basic residues, the early canonical alphabet was remarkably adaptive at supporting protein folding and explain why basic residues were only incorporated at a later stage of the alphabet evolution.

biophysics↗

bioRxiv2022

Experimental characterisation of de novoproteins and their unevolved random-sequencecounterparts

De novo gene emergence provides a route for new proteins to be formed from previously non-coding DNA. Proteins born in this way are considered random sequences, and typically assumed to lack defined structure. While it remains unclear how likely a de novo protein is to assume a soluble and stable tertiary structure, intersecting evidence from random-sequence and de novo-designed proteins suggests that native-like biophysical properties are abundant in sequence space. Taking putative de novo proteins identified in human and fly, we experimentally characterise a library of these sequences to assess their solubility and structure propensity. We compare this library to a set of synthetic random proteins with no evolutionary history. Bioin-formatic prediction suggests that de novo proteins may have remarkably similar distributions of biophysical properties to unevolved random sequences of a given length and amino acid composition. However, upon expression in vitro, de novo proteins exhibit higher solubility which is further induced by the DnaK chaperone system. We suggest that while synthetic ran-dom sequences are a useful proxy for de novo proteins in terms of structure propensity, de novo proteins may be better integrated in the cellular system given their higher solubility.

evolutionary biology↗

bioRxiv2021

Structured proteins are abundant in unevolved sequence space

Natural proteins represent numerous but tiny structure/function islands in a vast ocean of possible protein sequences, most of which has not been explored by either biological evolution or research. Recent studies have suggested this uncharted sequence space possesses surprisingly high structural propensity, but development of an understanding of this phenomenon has been awaiting a systematic high-throughput approach. Here, we designed, prepared, and characterized two combinatorial protein libraries consisting of randomized proteins, each 105 residues in length. The first library constructed proteins from the entire canonical alphabet of 20 amino acids. The second library used a subset of only 10 residues (A,S,D,G,L,I,P,T,E,V) that represent a consensus view of plausibly available amino acids through prebiotic chemistry. Our study shows that compact conformations resistant to proteolysis are (i) abundant (up to 40%) in random sequence space, (ii) independent of general Hsp70 chaperone system activity, and (iii) not granted solely by "late" and complex amino acid additions. The Hsp70 chaperone system effectively increases solubility and refoldability of the canonical alphabet but has only a minor impact on the "early" library. The early alphabet proteins are inherently more soluble and refoldable, possibly assisted by the cell-like environment in which these assays were performed. Our work indicates that both early and modern amino acids are predisposed to supporting protein structure (either in forms of oligomers or globular/molten globule structures) and that protein structure may not be a unique outcome of evolution.

synthetic biology↗

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↗