Biology subjects

Christodoulou, J.

Publications and source records attributed to Christodoulou, J..

At least 19 recordsLinked to original sources

bioRxiv2026

The initiation of de novo protein folding on the ribosome

How the earliest structure within the unfolded state is formed during biosynthesis on the ribosome and whether it has any consequences for downstream folding remain open questions. Here, we combine 15N paramagnetic relaxation enhancement NMR with all-atom molecular dynamics simulations to characterise the unfolded state of a folding-competent immunoglobulin-like domain on the ribosome at the cusp of folding initiation. We identify three structurally distinct sub-ensembles that differ in compaction and ribosome interactions. Non-native contacts, together with ribosome interactions, likely delay folding, yet their persistence alongside early native-like contacts within a sparsely populated compact sub-ensemble suggests they may also facilitate the formation of a co-translational folding nucleus, whose contacts overlap with those of the downstream intermediates. From these findings we infer a mechanistic model of de novo folding initiation during biosynthesis and, by linking the folding nucleus to downstream partially structured intermediates and the native state, provide a complete atomistic description of a co-translational folding pathway.

biophysics↗

bioRxiv2026

A flexible diet platform for the nutrigenomic screening of Drosophila disease models

Nutrient-gene interactions shape metabolic disease phenotypes, but systematic testing is limited by the effort required to produce defined diets. We developed a flexible protocol that assembles precisely defined synthetic diets for Drosophila melanogaster from individual stock solutions. Using this approach, we generated a rational array of 51 single-nutrient-varied diets and demonstrated that flexible and standard preparation methods produce comparable developmental timing, survival, adult body weight, and starvation resistance in wildtype flies. Applying the dietary array to a Drosophila model of isolated sulfite oxidase deficiency, a severe condition caused by impaired sulfur amino acid catabolism, revealed nutrient-specific effects on pupal survival and pupariation timing, including rescue by cysteine depletion and other amino acid modified diets. This platform provides a scalable in vivo framework for mapping genotype-specific nutritional responses across Drosophila disease models. Key pointsO_LIDeveloped a flexible platform for assembling precisely defined synthetic diets in Drosophila. C_LIO_LIGenerated a rational 51-diet array, enabling scalable nutrigenomic screening. C_LIO_LIFlexible and standard diet preparations yielded matching developmental and adult fitness outcomes. C_LIO_LIDietary array screening in a sulfite oxidase deficiency model recapitulated cysteine sensitivity and identified novel nutrient modifiers. C_LI

genetics↗

bioRxiv2025

Protein Dynamics at Different Timescales Unlock Access to Hidden Post-Translational Modification Sites

Post-translational modifications (PTMs) alter the proteome in response to intra- and extracellular signals, providing fundamental information processing in development, homeostasis and disease1-5. Here, we used proteome-wide structure predictions to investigate the structural context of protein modification sites and found nearly a fifth to be buried within proteins6. These cryptic sites within folded domains showed evolutionary conservation across species, as well as significantly less variability within the human population. We also found significantly more modification sites to be affected by recurrent cancer mutations and disease variants, with some variants appearing to mimic the modified or unmodified state, supporting their functionality and disease relevance. To understand how these buried sites become modified, we studied 23 phosphorylation sites across seven proteins using all-atom molecular dynamics simulations. The simulations revealed a range of mechanisms spanning broad timescales, ranging from picosecond fluctuations to seconds for proline isomerisation-gated phosphorylation sites. Notably, some sites remained fully buried in the native structure. Some conformational states with exposed cryptic sites resembled structures of co-translational protein folding intermediates formed on the ribosome7, indicating an interplay between protein biogenesis, folding, modifications, and signalling. We propose more broadly that protein conformational fluctuations may serve as structure-specific regulatory mechanisms by kinetically burying or exposing the unmodified, and in some cases even modified, sidechains, thereby expanding the functional versatility of proteins and enabling signal transduction across diverse timescales.

bioinformatics↗

bioRxiv2025

Visualisation of translating ribosomes reveals the earliest steps of protein misfolding in human disease

The majority of cellular proteins must adopt a particular three-dimensional structure for function1. However, protein folding is a perilous journey due to competing polypeptide misfolding events which result in inactive structures. In this study, we examine the earliest steps of protein misfolding during the biosynthesis of alpha-1-antitrypsin, a secreted plasma protein whose misfolding results in organ disease. Using human cells, we find that, like co-translational protein folding, misfolding, assembly and biosynthesis are interconnected processes. At the molecular level misfolding of alpha-1-antitrypsin is initiated by a molten globule-like folding intermediate formed cotranslationally on the ribosome. The ribosomal complexes subsequently form assemblies by recruiting released proteins, inducing translational arrest. Our data also reveal that a pharmacological chaperone modulates this process. The existence of co- and post-translational (mis)folding and assembly pathways reveals how some proteins form functional complexes, has implications for the pathogenesis of conformational diseases, and suggests novel therapeutic avenues.

biochemistry↗

bioRxiv2025

Structures of protein folding intermediates on the ribosome

The ribosome biases the conformations sampled by nascent polypeptide chains along folding pathways towards biologically active states. A hallmark of the co-translational folding (coTF) of many proteins are highly stable folding intermediates that are absent or only transiently populated off the ribosome, yet persist during translation well-beyond complete emergence of the domain from the ribosome exit tunnel. Intermediates are important for folding fidelity; however, their structures have remained elusive. Here, we have structurally characterised two coTF intermediates of an immunoglobulin-like domain by developing comprehensive 19F NMR analyses using chemical shifts, paramagnetic relaxation enhancement (PRE), and protein engineering. We integrated these experimental data with extensive molecular dynamics (MD) simulations to obtain atomistic structures of the folding intermediates on the ribosome. The resulting intermediate structures are distinguished by native-like folds initiated from either their N-or C-termini, and reveal parallel folding pathways, which are structurally conserved within the protein domain family, in contrast to their in vitro refolding mechanisms. By redirecting proteins to fold along hierarchical, parallel routes, the ribosome may promote efficient folding by avoiding kinetic traps, and regulate nascent chain assembly and targeting by auxiliary factors to maintain cellular proteostasis.

biophysics↗

bioRxiv2025

The ribosome directs nascent chains through two folding-dependent pathways

During their vectorial biosynthesis on the ribosome, elongating nascent polypeptide chains explore a range of conformational states towards their biologically functional structure. However, this high structural heterogeneity has limited their observation at high-resolution. Here, we have used an integrated structural biology approach to explore the structures of the multi-domain immunoglobulin-like FLN5-6 during its biosynthesis, capturing early folding through to native folding. We developed an in-silico purification approach for cryo-EM of ribosome-nascent chain complexes (RNCs), and integrated the resulting cryo-EM maps with NMR spectroscopy and atomistic molecular dynamics (MD) simulations to produce experimentally reweighted structural ensembles of RNC. The resulting atomistic structures reveal insights into the orientational heterogeneity of the nascent chain and its dynamic interactions with the ribosome. In particular, we find that two distinct pathways exist for nascent polypeptides in the exit tunnel vestibule, influenced by their stage of biosynthesis, folding conformational state and ribosomal RNA helices lining the tunnel. Our systematic analysis of the structures of nascent proteins translation-stalled at multiple time-points provides insights into how the ribosome dynamically modulates its pathway out of the exit tunnel to regulate its folding and accessibility for auxiliary factors of other co-translational events.

biophysics↗

bioRxiv2025

Long-range electrostatic forces govern how proteins fold on the ribosome

Protein biosynthesis and folding are tightly intertwined processes regulated by the ribosome and auxiliary factors. Nascent proteins can begin to fold on their parent ribosome but formation of the native state is often inhibited well beyond the emergence of the necessary residues from the exit tunnel. The dominant forces driving this apparent destabilisation have remained unclear. We investigate this here, combining NMR experiments and atomistic simulations of a folded nascent chain on and off the ribosome. While its native structure and internal dynamics remain unaltered, co-translational folding is disfavoured due to intermolecular electrostatic repulsion between the negatively charged ribosome surface and nascent protein. Partially folded intermediates are less destabilised, resulting in their high populations. Specifically, we show that the polypeptides net charge is the dominant factor determining nascent folding thermodynamics, with smaller contributions from charge distribution. Consequently, positively charged proteins can fold on the ribosome without populating stable intermediates. These findings reconcile conflicting observations of previous studies and establish the general physical principles underpinning de novo protein folding.

biophysics↗

bioRxiv2024

Amyloid forming human lysozyme intermediates are stabilised by non-native amide-π interactions

Mutational variants of human lysozyme cause a rare but fatal hereditary form of systemic amyloidosis by populating an intermediate state that self-assembles into amyloid fibrils. Despite its significance in lysozyme amyloidosis, the intermediate state has been recalcitrant to detailed structural investigation as it is only transiently and sparsely populated. Here, we investigated the intermediate state of a mutational variant of human lysozyme (I59T) using CEST and CPMG RD NMR at low pH. 15N CEST profiles probed the thermal unfolding of the native state into the denatured ensemble and revealed an additional state distinct from the two major states. Global fitting of 15N CEST and CPMG data provided kinetic and thermodynamic parameters for the exchange between all three states, characterising the intermediate state populated at 0.6%. 1H CEST data also confirmed the presence of the intermediate state displaying unusually high or low 1HN chemical shifts. To further investigate the structural details of the intermediate state we used molecular dynamics (MD) simulations, which recapitulated the experimentally observed folding pathway and free energy landscape. A high-energy intermediate state with a locally disordered {beta}-domain and C-helix was observed, revealing non-native hydrogen bonding and amide-{pi} interactions. These interactions account for the anomalous 1H chemical shifts and likely stabilise the transient intermediate state structure. Together, our NMR and MD data provide the first direct structural information on the intermediate state, offering insights into targeting lysozyme amyloidosis.

biophysics↗

bioRxiv2024

Rational design of 19F NMR labelling sites to probe protein structure and interactions

Proteins are investigated in increasingly more complex biological systems, where 19F NMR is proving highly advantageous due to its high gyromagnetic ratio and background-free spectra. Its application has, however, been hindered by limited chemical shift dispersions and an incomprehensive relationship between chemical shifts and protein structure. We exploit the sensitivity of 19F chemical shifts to ring currents by designing labels with direct contact to a native or engineered aromatic ring. Fifty protein variants predicted by AlphaFold and molecular dynamics simulations show 80-90% success rates and direct correlations of their experimental chemical shifts with the magnitude of the engineered ring current. Our method consequently improves the chemical shift dispersion and through simple 1D experiments enables structural analyses of alternative conformational states, including ribosome-bound folding intermediates, and in-cell measurements of thermodynamics and protein-protein interactions. Our strategy thus provides a simple and sensitive tool to extract residue contact restraints from chemical shifts for previously intractable systems.

biophysics↗

bioRxiv2024

Valine restriction extends survival in a Drosophila model of short-chain enoyl-CoA hydratase 1 (ECHS1) deficiency

Short-chain enoyl-CoA hydratase 1 deficiency (ECHS1D) is a rare genetic disorder caused by biallelic pathogenic variants in the ECHS1 gene. ECHS1D is characterised by severe neurological and physical impairment that often leads to childhood mortality. Therapies such as protein and single nutrient-restricted diets show poor efficacy, whereas development of new treatments is hindered by the low prevalence of the disorder and a lack of model systems for treatment testing. Here we report on the establishment of a Drosophila model of ECHS1D. Flies carrying mutations in Echs1 (CG6543) were characterised for their physical and metabolic phenotypes, and dietary intervention to improve fly model health was explored. The Echs1 null larvae recapitulated human ECHS1D phenotypes including elevated biomarkers (S-(2-carboxypropyl)cysteamine and 2,3-dihydroxy-2-methylbutyric acid), poor motor behaviour and early mortality, and could be rescued by expression of a human ECHS1 transgene. We observed that both restriction of valine in isolation, or all branched-chain amino acids (BCAAs - leucine, isoleucine, and valine) together, extended larval survival, supporting the idea that reducing BCAA pathway catabolic flux is beneficial in this disorder. Further, metabolic profiling revealed substantial changes to carbohydrate metabolism, suggesting that Echs1 loss causes widespread metabolic dysregulation beyond valine metabolism. The similarities between Drosophila and human ECHS1D suggest that the fly model is a valuable animal system in which to explore mechanisms of pathogenesis and novel treatment options for this disorder.

genetics↗

bioRxiv2023

CryoENsemble - a Bayesian approach for reweighting biomolecular structural ensembles using heterogeneous cryo-EM maps

Cryogenic electron microscopy (cryo-EM) has emerged as a central tool for the determination of structures of complex biological molecules. Accurately characterising the dynamics of such systems, however, remains a challenge. To address this, we introduce cryoENsemble, a method that applies Bayesian reweighing to conformational ensembles derived from molecular dynamics simulations to improve their agreement with cryo-EM data and extract dynamics information. We illustrate the use of cryoENsemble to determine the dynamics of the ribosome-bound state of the co-translational chaperone trigger factor (TF). We also show that cryoENsemble can assist with the interpretation of low-resolution, noisy or unaccounted regions of cryo-EM maps. Notably, we are able to link an unaccounted part of the cryo-EM map to the presence of another protein (methionine aminopeptidase, or MetAP), rather than to the dynamics of TF, and model its TF-bound state. Based on these results, cryoENsemble is expected to find use for challenging heterogeneous cryo-EM maps for various biomolecular systems, especially those encompassing dynamic elements.

biophysics↗

bioRxiv2023

Thermodynamic profiles for co-translational trigger factor function

Molecular chaperones are central to the maintenance of proteostasis in living cells. A key member of this protein family is trigger factor (TF), which acts throughout the protein lifecycle and has a ubiquitous role as the first chaperone encountered by proteins during synthesis. However, our understanding of how TF achieves favourable interactions with such a diverse substrate base remains limited. Here, we use microfluidics to reveal the thermodynamic determinants of this process. We find that TF binding to empty 70S ribosomes is enthalpydriven, with micromolar affinity, while nanomolar affinity is achieved through a favourable entropic contribution for both intrinsically disordered and folding competent nascent chains. These findings suggest a general mechanism for co-translational TF function, which relies on occupation of the exposed TF substrate-binding groove, rather than specific complementarity between chaperone and RNC. These insights add to our wider understanding of how proteins can achieve broad substrate specificity.

biophysics↗

bioRxiv2022

Quantifying constraint in human mitochondrial DNA

Mitochondrial DNA (mtDNA) has an important, yet often overlooked, role in health and disease. Constraint models quantify the removal of deleterious variation from the population by selection, representing a powerful tool for identifying genetic variation underlying human phenotypes1-4. However, a constraint model for the mtDNA has not been developed, due to its unique features. Here we describe the development of a mitochondrial constraint model and its application to the Genome Aggregation Database (gnomAD), a large-scale population dataset reporting mtDNA variation across 56,434 humans5. Our results demonstrate strong depletion of expected variation, suggesting most deleterious mtDNA variants remain undiscovered. To aid their identification, we compute constraint metrics for every mitochondrial protein, tRNA, and rRNA gene, revealing a spectrum of intolerance to variation. We characterize the most constrained regions within genes via regional constraint, and positions across the entire mtDNA via local constraint, showing their enrichment in pathogenic variation and functionally critical sites, including topological clustering in 3D protein and RNA structures. Notably, we identify constraint at often overlooked sites, such as rRNAs and non-coding regions. Lastly, we demonstrate how these metrics can improve the discovery of mtDNA variation underlying rare and common human phenotypes.

genetics↗

bioRxiv2021

An intrinsic temporal order of c-Jun N-terminal phosphorylation regulates its activity by orchestrating co-factor recruitment

Protein phosphorylation is a major regulatory mechanism of cellular signalling. The c-Jun proto-oncoprotein is phosphorylated at four residues within its transactivation domain (TAD) by the JNK family kinases, but the functional significance of c-Jun multisite phosphorylation has remained elusive. Here we show that c-Jun phosphorylation by JNK exhibits a defined temporal kinetics, with serine63 and serine73 being phosphorylated more rapidly than threonine91 and threonine93. We identified the positioning of the phosphorylation sites relative to the kinase docking motif, and their primary sequence, as the main factors controlling phosphorylation kinetics. Functional analysis revealed three c-Jun phosphorylation states: unphosphorylated c-Jun recruits the Mbd3 repressor, serine63/73 doubly-phosphorylated c-Jun binds to the Tcf4 co-activator, whereas the fully phosphorylated form disfavours Tcf4 binding attenuating JNK signalling. Thus, c-Jun phosphorylation encodes multiple functional states that drive a complex signalling response from a single JNK input.

biochemistry↗

bioRxiv2021

Analysis of Conformational Exchange Processes using Methyl-TROSY-Based Hahn Echo Measurements of Quadruple-Quantum Relaxation

Transverse nuclear spin relaxation can be a sensitive probe of chemical exchange on timescales on the order of microseconds to milliseconds. Here we present an experiment for the simultaneous measurement of the relaxation rates of two four-spin transitions in selectively protonated methyl groups within perdeuterated biomacromolecules, alongside control experiments for measurement of 1H and 13C chemical shift anisotropies. We show that analysis of the static magnetic field dependence of zero-, double- and quadruple-quantum Hahn echo relaxation rates provides a robust indication of chemical exchange and determines the signed relative magnitudes of proton and carbon chemical shift differences between ground and excited states. The analysis can be combined with CPMG relaxation dispersion measurements to provide improved precision, particularly in the determination of 1H chemical shift differences.

biophysics↗

bioRxiv2020

Optimal design of adaptively sampled NMR experiments for measurement of methyl group dynamics with application to a ribosome-nascent chain complex

NMR measurements of cross-correlated nuclear spin relaxation provide powerful probes of polypeptide dynamics and rotational diffusion, free from contributions due to chemical exchange or interactions with external spins. Here, we report on the development of a sensitivity-optimized pulse sequence for the measurement of cross-correlated relaxation in methyl spin systems by analysis of the differential relaxation of transitions within the 13C multiplet. We describe the application of optimal design theory to implement a real-time on-the-fly adaptive sampling scheme that maximizes the accuracy of the measured rate constants. The increase in sensitivity obtained using this approach enables, for the first time, quantitative measurements of rotational diffusion within folded states of translationally-arrested ribosome-nascent chain complexes of the FLN5 filamin domain, and can be used to place strong limits on interactions between the domain and the ribosome surface.

biophysics↗

bioRxiv2019

Loss of function variants in PCYT1A causing spondylometaphyseal dysplasia with cone/rod dystrophy have broad consequences on lipid metabolism, chondrocyte differentiation, and lipid droplet formation.

AbstractSpondylometaphyseal dysplasia with cone-rod dystrophy (SMD-CRD) is a rare autosomal recessive disorder of the skeleton and the retina caused by biallelic variants in PCYT1A, encoding the nuclear enzyme CTP:phosphocholine cytidylyltransferase (CCT), which catalyzes the rate-limiting step in phosphatidylcholine (PC) biosynthesis by the Kennedy pathway. As a first step in understanding the consequences of PCYT1A variants on SMD-CRD pathophysiology, we generated and characterized a series of cellular models for SMD-CRD, including CRISPR-edited PCYT1A-null HEK293 and ATDC5 cell lines. Immunoblot and PC synthesis assays of cultured skin fibroblasts from SMD-CRD patient cell lines revealed patient genotype-specific reductions in CCT steady state levels (10-75% of wild-type) and choline incorporation into PC (22-54% of wild-type). While PCYT1A-null HEK293 cells exhibited fewer and larger lipid droplets in response to oleate loading than their wild-type counterparts, SMD-CRD patient fibroblasts (p.Ser323Argfs*38 homozygotes) failed to show significant differences in lipid droplet numbers or sizes as compared to controls. Lipid droplet phenotypes in PCYT1A-null HEK293 cells were rescued by transfection with wild-type, p.Ala99Val, and p.Tyr240His human PCYT1A cDNAs. While both edited cellular models had normal morphology and proliferation rates compared to unedited controls, Pcyt1a-null ATDC5 cells demonstrated accelerated rates of chondrocyte differentiation as compared to their wild-type counterparts. Lipidomics revealed changes in 75-200 lipid levels in PCYT1A-null HEK293 and ATDC5 cells or in SMD-CRD patient fibroblasts as compared to wild-type controls. The specific lipids altered and extent of change varied by cell type. Importantly, both PCYT1A-null HEK293 cells and SMD-CRD patient fibroblast cell lines had decreased phosphatidylcholine:phosphatidylethanolamine (PC:PE) ratios and decreased levels of several lysophosphatidylcholine (LPC) species as compared to wild-type controls, suggesting compensatory PC production through increased LPC remodeling by LPCAT or decreased conversion of PC to LPC by phospholipase A2. Our results show that all tested PCYT1A alleles associated with SMD-CRD are hypomorphic and suggest involvement of PCYT1A in chondrocyte differentiation, PC:PE ratio maintenance and LPC metabolism, and lipid droplet formation. Author SummaryRare genetic disorders can reveal the function of genes on an organismal scale. When normal gene activity is lost, patients can experience a range of symptoms, often dependent on the residual activity of the encoded protein. Rare variants in the gene PCYT1A can cause multiple inherited disorders, including a disorder of the skeleton and the retina characterized by short stature, bone abnormalities, and blindness. PCYT1A is required for normal cellular function, particularly lipid metabolism, but the role of this gene in human disease is still poorly understood. To determine consequences of genetic variants in patients with this disorder, we made and studied a series of cellular models, including cells cultured from patients and CRISPR-edited cell lines lacking normal copies of PCYT1A. Here we show that patient variants lead to reduced PCYT1A expression and/or function and have adverse consequences on cell biology and lipid metabolism that are often cell-type specific. This work advances understanding of the role of lipid metabolism in skeletal and eye development.

genetics↗

bioRxiv2019

Rapid diagnosis of SCA36 in a three-generation family using short-read whole genome sequencing data

BackgroundSpinocerebellar ataxias (SCA) are often caused by expansions of short tandem repeats (STRs). Recent methodological advances have made repeat expansion (RE) detection with whole genome sequencing (WGS) feasible. ObjectivesTo determine the genetic basis of ataxia in a multigenerational Australian pedigree, with autosomal dominant inheritance. Methods and ResultsWGS was performed on three affected relatives. The sequence data was screened for known pathogenic REs using two repeat expansion detection tools: exSTRa and ExpansionHunter. This screen provided a clear and rapid diagnosis (<five days from receiving the sequencing data) of SCA36, a rare form of ataxia caused by an intronic GGCCTG RE in NOP56. Conclusionsthe that diagnosis of rare ataxias caused by REs is highly feasible and cost effective with WGS. We propose that WGS be implemented as the frontline, cost effective methodology for molecular testing of individuals with a clinical diagnosis of ataxia.

genomics↗