Biology subjects

Cabrita, L. D.

Publications and source records attributed to Cabrita, L. D..

7 recordsLinked to original sources

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↗

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↗

bioRxiv2018

Developing NMR methods for macromolecular machines: Measurement of residual dipolar couplings to probe dynamical regions of the ribosome

We describe an NMR approach based on the measurement of residual dipolar couplings (RDCs) to probe the structural and motional properties of the dynamic regions of the ribosome. Alignment of intact 70S ribosomes in filamentous bacteriophage enabled measurement of RDCs in the stalk protein bL12, and this information was used to refine a 3D structure of its C-terminal domain (CTD). Orientational constraints on the CTD alignment arising from the semiflexible linker sequence were further probed by a paramagnetic alignment strategy, and provided direct experimental validation of a structural ensemble previously derived from SAXS and NMR relaxation measurements. Our results demonstrate the prospect of better characterising dynamical and functional regions of more challenging macromolecular machines and systems, for example ribosome-nascent chain complexes.

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↗