Biology subjects

Chan, S. H. S.

Publications and source records attributed to Chan, S. H. S..

6 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↗

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

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↗