Biology subjects

Wlodarski, T.

Publications and source records attributed to Wlodarski, T..

5 recordsLinked to original sources

bioRxiv2025

Evolution of the ribosomal exit tunnel through the eyes of the nascent chain

The exit tunnel is a universally conserved feature of the ribosomal large subunit that directs the nascent polypeptide chain into the cellular environment and is involved in co-translational folding, stalling, and antibiotic binding. While recent advances in cryo-EM have revealed organism-specific variations in ribosome structure, tunnel definition and comparative analyses have largely relied on pure geometric algorithms that often approximate the tunnel as a static, single tube with varying radius. Here, we present a functional, nascent-chain-centric characterisation of the exit tunnel across the tree of life, derived from all-atom molecular dynamics simulations using the most complete set of 55 distinct cytoplasmic ribosome structures. By mapping the steric accessibility through the "eyes" of the nascent chain (NC) at five different stages of translation, we reveal a topological and stage-dependent complexity invisible to static geometric approaches, demonstrating how tunnel accessibility dynamically changes during biosynthesis. We identify transiently accessible, bacteria-specific lateral branches ("side tunnels") that effectively expand the functional volume of the tunnel but may also serve as kinetic traps for the nascent chain. In contrast, we show that archaeal and eukaryotic ribosomes have structurally occluded these cavities by incorporating the eL39 protein. These evolutionary "plugs" seal the tunnel wall and decrease its functional width. Collectively, our results demonstrate that the ribosome exit tunnel is not a simple linear tube, but a branched, lineage-specific landscape where accessibility is temporally gated by nascent chain length. This functional definition provides a new framework for understanding how ribosomal architecture modulates the early stages of protein biogenesis.

biophysics↗

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↗

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↗

bioRxiv2019

Visualising nascent chain dynamics at the ribosome exit tunnel by cryo-electron microscopy

Ribosomes maintain a healthy cellular proteome by synthesising proteins. The nascent chain (NC), emerges into the cellular milieu via the ribosomal exit tunnel, which is an active component that regulates the NC passage. How the NC dynamics at the exit tunnel affect NC folding remains to be an important question, the answer on which has strong implications to medicine. Here, we report high-resolution cryo-EM maps of ribosome nascent-chain complexes (RNCs) displaying distinct steps during biosynthesis. These RNC structures reveal a range of pathways adopted by the NC. The most pronounced diversity in the NC trajectories were found in the vestibule region. Rearrangements of several ribosomal components further suggest that these elements may actively monitor the emerging NC during translation. The ribosome-NC contacts within the vestibule define these NC pathways and modulate position of a folded immunoglobulin domain outside the ribosome.

biophysics↗

bioRxiv2017

Systematic mapping of the free energy landscapes of a growing immunoglobulin domain identifies a kinetic intermediate associated with co-translational proline isomerization

Co-translational folding is a fundamental molecular process that ensures efficient protein biosynthesis and minimizes the wasteful or hazardous formation of misfolded states. However, the complexity of this process makes it extremely challenging to obtain structural characterizations of co-translational folding pathways. Here we contrast observations in translationally-arrested nascent chains with those of a systematic C-terminal truncation strategy. We create a detailed description of chain length-dependent free energy landscapes associated with folding of the FLN5 filamin domain, in isolation and on the ribosome. By using this approach we identify and characterize two folding intermediates, including a partially folded intermediate associated with the isomerization of a conserved proline residue, which, together with measurements of folding kinetics, raises the prospect that neighboring unfolded domains might accumulate during biosynthesis. We develop a simple model to quantify the risk of misfolding in this situation, and show that catalysis of folding by peptidyl-prolyl isomerases is essential to eliminate this hazard.

biophysics↗