Arabidopsis thaliana Cyclic Nucleotide-Gated Channel 19 is involved in root extracellular ATP and Pep1 signalling
New Phytol. 2025 Oct 5. doi: 10.1111/nph.70624. Online ahead of print.
NO ABSTRACT
PMID:41047608 | DOI:10.1111/nph.70624
The potential for biased signalling in the P2Y receptor family of GPCRs
Br J Pharmacol. 2025 Oct 3. doi: 10.1111/bph.70213. Online ahead of print.
ABSTRACT
The purinergic receptor family is primarily activated by nucleotides, and contains members of both the G protein coupled-receptor (GPCR) superfamily (P1 and P2Y) and ligand-gated ion channels (P2X). The P2Y receptors are widely expressed in the human body, and given the ubiquitous nature of nucleotides, purinergic signalling is involved with a plethora of molecular physiological functions. The widespread nature of P2Y receptors make them a viable therapeutic target, but with the negative risk of on-target side effects. Some of the side effects associated with P1 and P2Y receptors arise due to the pleiotropic nature of many GPCRs, because a singular GPCR can activate several G proteins, as well as recruit β-arrestins. The utilisation of ligands that exhibit downstream pathway-specific activation, also known as biased signalling, at the P2Y receptors could circumvent these issues. This review will cover the signalling nature and impact of the P2Y family, with an emphasis on individual activity patterns of the P2Y receptors. This review also discusses current literature on the development of biased ligands for these receptors, with an aim to highlight the most beneficial targets and outcomes.
PMID:41041819 | DOI:10.1111/bph.70213
Split NeissLock with Spy-Acceleration Arms Mammalian Proteins for Anhydride-Mediated Cell Ligation
ACS Chem Biol. 2025 Sep 15. doi: 10.1021/acschembio.5c00515. Online ahead of print.
ABSTRACT
Reactive functional groups may be incorporated into proteins or may emerge from natural amino acids in exceptional architectures. Anhydride formation is triggered by calcium in the self-processing module (SPM) of Neisseria meningitidis FrpC, which we previously engineered for "NeissLock" ligation to an unmodified target protein. Here, we explored bacterial diversity, discovering a related module with ultrafast anhydride formation. We dissected this swift SPM to generate a split NeissLock system, providing a second layer of control of anhydride generation: first mixing N- and C-terminal NeissLock moieties and second adding millimolar amounts of calcium. Split NeissLock generated a minimal fusion tag, permitting binder expression in mammalian cells with complex post-translational modifications and avoiding self-cleavage while transiting the calcium-rich secretory pathway. Employing spontaneous amidation between SpyTag003 and SpyCatcher003, we dramatically accelerated split NeissLock reconstitution, allowing a rapid high-yield reaction to naturally occurring targets. We established a specific covalent reaction to endogenous Epidermal Growth Factor Receptor using split NeissLock via Transforming Growth Factor-α secreted from mammalian cells. Modular ligation was demonstrated on living cells through site-specific coupling of the clot-busting enzyme tissue plasminogen activator or a computationally designed cytokine. Split NeissLock provides a modular architecture to generate highly reactive functionality, with inducibility and simple genetic encoding for enhanced cellular modification.
PMID:40947984 | DOI:10.1021/acschembio.5c00515
Defining breast epithelial cell types in the single-cell era
Dev Cell. 2025 Sep 8;60(17):2218-2236. doi: 10.1016/j.devcel.2025.06.032.
ABSTRACT
Single-cell studies on breast tissue have contributed to a change in our understanding of breast epithelial diversity that has, in turn, precipitated a lack of consensus on breast cell types. The confusion surrounding this issue highlights a possible challenge for advancing breast atlas efforts. In this perspective, we present our consensus on the identities, properties, and naming conventions for breast epithelial cell types and propose goals for future atlas endeavors. Our proposals and their underlying thought processes aim to catalyze the adoption of a shared model for this tissue and to serve as guidance for other investigators facing similar challenges.
PMID:40925326 | DOI:10.1016/j.devcel.2025.06.032
Using modified mRNA for cardiomyocyte proliferation and cardiac genetic disease modelling and treatment
Biochem Soc Trans. 2025 Sep 4:BST20243001. doi: 10.1042/BST20243001. Online ahead of print.
ABSTRACT
Heart failure (HF) is a leading cause of death worldwide and the associated mortality and socioeconomic burden is predicted to worsen. Current therapies for HF focus on managing the causes and symptoms; however, these current treatment options are unable to reverse heart muscle degeneration, with heart transplantation the only cure. The ability to re-muscularise the heart represents a significant unmet clinical need. Although numerous biological pathways driving re-muscularisation have been identified, delivery of therapeutic factors is challenging. Modified mRNA (modRNA) is synthetic mRNA with greater gene packaging capacity, low immunogenic response and allows transient but robust protein expression. In this mini-review, we highlight the emerging discoveries surrounding the application of modRNA in the cardiovascular field. Specifically, we focus on different examples illustrating how modRNA delivery post-myocardial infarction can drive cardiomyocyte proliferation and achieve cardiac regeneration. In addition, we demonstrate how modRNA is being used for protein replacement and Cas delivery for both modelling and therapeutic studies focussed on genetic cardiac diseases. For these applications, in particular Cas delivery, the transient nature of modRNA overexpression is a beneficial property with reduced side effects compared with other modalities. Finally, we preview some of the roadblocks limiting the clinical translation of modRNA and avenues being explored to overcome these. In summary, the flexibility of modRNA combined with its improved safety profile provides a gene overexpression tool capable of integration into all steps of the preclinical and clinical therapeutic pipeline enabling the discovery of improved treatments for HF.
PMID:40905915 | DOI:10.1042/BST20243001
Development of D-box peptides to inhibit the anaphase-promoting complex/cyclosome
Elife. 2025 Sep 1;14:RP104238. doi: 10.7554/eLife.104238.
ABSTRACT
E3 ubiquitin ligases engage their substrates via 'degrons' - short linear motifs typically located within intrinsically disordered regions of substrates. As these enzymes are large, multi-subunit complexes that generally lack natural small-molecule ligands and are difficult to inhibit via conventional means, alternative strategies are needed to target them in diseases, and peptide-based inhibitors derived from degrons represent a promising approach. Here we explore peptide inhibitors of Cdc20, a substrate-recognition subunit and activator of the E3 ubiquitin ligase the anaphase-promoting complex/cyclosome (APC/C) that is essential in mitosis and consequently of interest as an anti-cancer target. APC/C engages substrates via degrons that include the 'destruction box' (D-box) motif. We used a rational design approach to construct binders containing unnatural amino acids aimed at better filling a hydrophobic pocket that contributes to the D-box binding site on the surface of Cdc20. We confirmed binding by thermal-shift assays and surface plasmon resonance and determined the structures of a number of the Cdc20-peptide complexes. Using a cellular thermal shift assay, we confirmed that the D-box peptides also bind to and stabilise Cdc20 in the cell. We found that the D-box peptides inhibit ubiquitination activity of APC/CCdc20 and are more potent than the small-molecule inhibitor Apcin. Lastly, these peptides function as portable degrons capable of driving the degradation of a fused fluorescent protein. Interestingly, we find that although inhibitory activity of the peptides correlates with Cdc20-binding affinity, degradation efficacy does not, which may be due to the complex nature of APC/C regulation and effects of degron binding of subunit recruitment and conformational changes. Our study lays the groundwork for the further development of these peptides as molecular therapeutics for blocking APC/C as well as potentially for harnessing the APC/C for targeted protein degradation.
PMID:40888475 | PMC:PMC12401543 | DOI:10.7554/eLife.104238
Cap-independent co-expression of dsRNA-sensing and NF-κB pathway inhibitors enables controllable self-amplifying RNA expression with reduced immunotoxicity
Elife. 2025 Aug 29;14:RP105978. doi: 10.7554/eLife.105978.
ABSTRACT
Self-amplifying RNA (saRNA) holds promise for durable therapeutic gene expression, but its broader utility beyond vaccines is limited by potent innate immune responses triggered during replication. These responses shut down translation, induce cytotoxicity, degrade host mRNAs, and drive cytokine production. While exogenous immunosuppressants can blunt these effects, they complicate treatment and risk systemic side effects. To address this, we engineered 'immune-evasive saRNA' that intrinsically suppresses the innate immune pathways triggered by its own replication. This strategy leverages cap-independent translation to co-express a suite of inhibitors from a single saRNA transcript, targeting key innate immune pathways, including protein kinase R (PKR), oligoadenylate synthase (OAS)/RNase L, and nuclear factor-κB (NF-κB). In primary mouse fibroblast-like synoviocytes, a cell type central to the pathology of joint diseases, immune-evasive saRNA enables sustained transgene expression without external immunosuppressants, substantially reducing cytotoxicity and antiviral cytokine secretion. Crucially, this system offers both concentration-dependent control of expression and on-demand termination via a small-molecule antiviral. Together, these findings establish a framework for developing saRNA therapeutics with an improved tolerability profile that can be switched off once therapeutic outcomes are met, offering a path toward a controllable gene expression platform that fills the therapeutic gap between the transience of mRNA and the permanence of viral vectors.
PMID:40879330 | PMC:PMC12396818 | DOI:10.7554/eLife.105978
Receptor Activity-Modifying Protein 3 enhances GLP-1-mediated Insulin Secretion
bioRxiv [Preprint]. 2025 Aug 13:2025.01.24.634724. doi: 10.1101/2025.01.24.634724.
ABSTRACT
The targeting of the Glucagon-like peptide-1 receptor (GLP-1R) for diabetes and obesity is not a novel strategy, with recent therapeutics showing efficacy in weight loss and glycaemic control. However, they are also associated with side effects, including gastrointestinal disruptions and pancreatitis. Developing agonists with different signalling profiles, or that exert some tissue selectivity can circumvent these on-target, unwanted effects. Receptor activity-modifying proteins (RAMPs) offer the potential to do both, through modulation of agonist binding and signalling, as well as surface expression. The GLP-1R was found to interact with RAMP3, with the heterodimer able to bind agonist at the cell surface. RAMP3 expression biased the receptor towards Ca2+ mobilisation, away from the canonical cAMP-driven signalling. When examining G protein coupling, the interaction with RAMP3 reduced activation of the cognate Gαs but increased secondary couplings to Gαq and Gαi. These increased couplings led to an elevation in glucose-stimulated insulin secretion when cells overexpressing RAMP3 were stimulated with GLP-1. A reciprocal effect was observed when looking at reduced expression of endogenous RAMP3, with a loss of sensitivity to GLP-1 in both glucose and insulin tolerance tests in a Ramp3 knockout mouse. The effects of this interaction can then inform selection of models and peptide design when targeting this receptor for therapeutic intervention.
PMID:40832355 | PMC:PMC12363685 | DOI:10.1101/2025.01.24.634724
Receptor Activity-Modifying Protein 3 enhances GLP-1-mediated Insulin Secretion
J Biol Chem. 2025 Aug 18:110604. doi: 10.1016/j.jbc.2025.110604. Online ahead of print.
ABSTRACT
The targeting of the Glucagon-like peptide-1 receptor (GLP-1R) for diabetes and obesity is not a novel strategy, with recent therapeutics showing efficacy in weight loss and glycaemic control. However, they are also associated with side effects, including gastrointestinal disruptions and pancreatitis. Developing agonists with different signalling profiles, or that exert some tissue selectivity can circumvent these on-target, unwanted effects. Receptor activity-modifying proteins (RAMPs) offer the potential to do both, through modulation of agonist binding and signalling, as well as surface expression. The GLP-1R was found to interact with RAMP3, with the heterodimer able to bind agonist at the cell surface. RAMP3 expression biased the receptor towards Ca2+ mobilisation, away from the canonical cAMP-driven signalling. When examining G protein coupling, the interaction with RAMP3 reduced activation of the cognate Gαs but increased secondary couplings to Gαq and Gαi. These increased couplings led to an elevation in glucose-stimulated insulin secretion when cells overexpressing RAMP3 were stimulated with GLP-1. A reciprocal effect was observed when looking at reduced expression of endogenous RAMP3, with a loss of sensitivity to GLP-1 in both glucose and insulin tolerance tests in a Ramp3 knockout mouse. The effects of this interaction can then inform selection of models and peptide design when targeting this receptor for therapeutic intervention.
PMID:40835007 | DOI:10.1016/j.jbc.2025.110604
Fluorescent GLP1R/GIPR dual agonist probes reveal cell targets in the pancreas and brain
Nat Metab. 2025 Aug 19. doi: 10.1038/s42255-025-01342-6. Online ahead of print.
ABSTRACT
Dual agonists targeting glucagon-like peptide-1 receptor (GLP1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR) are breakthrough treatments for patients with type 2 diabetes and obesity. Compared to GLP1R agonists, dual agonists show superior efficacy for glucose lowering and weight reduction. However, delineation of dual agonist cell targets remains challenging. Here, we develop and test daLUXendin and daLUXendin+, non-lipidated and lipidated fluorescent GLP1R/GIPR dual agonist probes, and use them to visualize cellular targets. daLUXendins are potent GLP1R/GIPR dual agonists that advantageously show less functional selectivity for mouse GLP1R over mouse GIPR. daLUXendins label rodent and human pancreatic islet cells, with a signal intensity of β cells > α cells = δ cells. Systemic administration of daLUXendin strongly labels GLP1R+ and GIPR+ neurons in circumventricular organs characterized by an incomplete blood-brain barrier but does not penetrate the brain beyond labelling seen with single (ant)agonists. At the single-molecule level, daLUXendin targets endogenous GLP1R-GIPR nanodomains, which differ in organization and composition from those targeted by a single agonist. daLUXendins reveal dual agonist targets in the pancreas and brain and exclude a role for brain penetration in determining the superior efficacy of dual agonists, shedding new light on different modes of action of dual agonists versus single agonists.
PMID:40830598 | DOI:10.1038/s42255-025-01342-6
IL-17 Stimulates Sensory Neurons and Sensitises Colonic Afferents to Noxious Stimuli in a PI3K Dependent Manner in Male Mice
J Neurochem. 2025 Aug;169(8):e70191. doi: 10.1111/jnc.70191.
ABSTRACT
Managing visceral pain associated with gastrointestinal (GI) disease remains a significant challenge due to the gut-related side effects and contraindicated use of many commonly used painkillers in people with inflammatory bowel disease (IBD). Consequently, it is crucial to deepen our understanding of the mediators and mechanisms underlying inflammatory pain in people with IBD. To do this, we compared bulk RNA sequencing data from colonic biopsy samples from people with IBD with single-cell RNA sequencing data from colon-projecting dorsal root ganglion (DRG) neurons in mice to generate an interactome of putative pro-nociceptive cytokine signalling pathways. This in silico analysis revealed a 10-fold increase in IL17A expression in samples from people with ulcerative colitis (UC) alongside marked co-expression of Il17ra with Trpv1 in colon-projecting DRG neurons in mice, highlighting a likely role for interleukin-17 (IL-17) in colonic nociceptor signalling in people with UC. In support of this, Ca2+ imaging studies demonstrated that IL-17 stimulates DRG sensory neurons co-sensitive to capsaicin in male and female mice, with a similar proportion responding in neuron-enriched cultures generated by magnetic-activated cell sorting, thus confirming that IL-17 directly activates DRG neurons. IL-17-evoked Ca2+ signals were attenuated by TRPV1 inhibition, consistent with nociceptor activation, and blocked by inhibition of phosphoinositide 3-kinase (PI3K) activity, consistent with the known role for PI3K as a downstream effector of IL-17 receptor signalling. In keeping with these observations, IL-17 enhanced colonic afferent responses to colorectal distension at noxious distension pressures in male mice, an effect also blocked by PI3K inhibition. Overall, these findings demonstrate a pro-nociceptive effect of IL-17 in the GI tract, thus highlighting the potential utility of IL-17-targeting therapies to reduce pain in people with UC.
PMID:40827457 | DOI:10.1111/jnc.70191
Small Molecule Activators of Protein Phosphatase 2A Exert Global Stabilising Effects on the Scaffold PR65
bioRxiv [Preprint]. 2025 Jul 29:2025.07.24.666388. doi: 10.1101/2025.07.24.666388.
ABSTRACT
Protein phosphatase 2A (PP2A), an important therapeutic target, comprises a scaffold subunit PR65 composed of 15 HEAT (Huntingtin/elongation/A-subunit/TOR1) repeats, a catalytic subunit, and one of many different regulatory subunits that enable binding to specific substrates. Recently, small molecule activators of PP2A (SMAPs) were identified, although their mechanisms of action have not been fully defined. Here we explore the interaction of PR65 with two SMAPs, ATUX-8385 and the non-functional DBK-776, using single-molecule optical tweezers, ensemble methods, and computational analysis. In the absence of SMAP, PR65 shows multiple unfolding and refolding transitions, and the force-extension profiles are very heterogeneous with evidence of misfolding. Similar heterogeneity has been observed for chemical-induced unfolding of tandem-repeat proteins like PR65, a consequence of the internal symmetry of the repeat architecture. In the presence of ATUX-8385, higher unfolding and refolding forces are observed globally, and there is less misfolding, suggesting that ATUX-8385 acts like a pharmacological chaperone. In contrast, DBK-766-binding induces higher unfolding forces for only a few repeats of PR65, suggestive of a more localised effect; moreover, subsequent stretch-relax cycles show that PR65 is irreversibly locked in the unfolded state. Docking and molecular dynamics simulations provide additional insights how SMAP binding modulates PR65 structure and function.
PMID:40766424 | PMC:PMC12324213 | DOI:10.1101/2025.07.24.666388
Artemin sensitises mouse (Mus musculus) and naked mole-rat (Heterocephalus glaber) sensory neurons in vitro
J Comp Physiol A Neuroethol Sens Neural Behav Physiol. 2025 Jul 24. doi: 10.1007/s00359-025-01752-7. Online ahead of print.
ABSTRACT
The naked mole-rat (NMR, Heterocephalus glaber) is a subterranean rodent that exhibits a range of unusual physiological traits, including diminished inflammatory pain. For example, nerve growth factor (NGF), a key inflammatory mediator, fails to induce sensitization of sensory neurons and thermal hyperalgesia in NMRs. This lack of NGF-induced neuronal sensitization and thermal hyperalgesia results from hypofunctional signaling of the NGF receptor, tropomyosin receptor kinase A (TrkA). Like NGF-TrkA signaling, the neurotrophic factor artemin, a member of the glial cell line-derived neurotrophic factor (GDNF) family, is implicated in mediating inflammatory pain through its receptor, GDNF family receptor α3 (GFRα3), which is expressed by a subset of dorsal root ganglia (DRG) sensory neurons. Here we investigated GFRα3 expression in DRG neurons of mice and NMRs, as well as measuring the impact of artemin on DRG sensory neuron function in both species in vitro. Using immunohistochemistry, we observed a similar abundance of GFRα3 in mouse and NMR DRG sensory neurons, high coexpression with the transient receptor potential vanilloid 1 (TRPV1) ion channel suggesting that these neurons are nociceptive neurons. Using in vitro electrophysiology to record from cultured DRG sensory neurons, we observed that artemin induced depolarization of the resting membrane potential and decreased the rheobase in both species, as well as diminishing the degree of TRPV1 desensitization to multiple capsaicin stimuli. Overall, results indicate that artemin similarly sensitizes sensory neurons in both mice and NMRs, future in vivo studies being required to confirm if the conserved in vitro sensitization also occurs in vivo.
PMID:40705105 | DOI:10.1007/s00359-025-01752-7
The osteoarthritis associated sphingolipid sphingomyelin 34:1 causes inflammatory pain in mice
Osteoarthritis Cartilage. 2025 Jul 19:S1063-4584(25)01091-X. doi: 10.1016/j.joca.2025.07.011. Online ahead of print.
ABSTRACT
OBJECTIVE: Osteoarthritis (OA) has a multifactorial pathogenesis, pain being the main symptom driving clinical decision making. Dysregulation of multiple mediators occurs in OA, the roles of many remaining to be identified. In dogs and humans with OA, synovial fluid lipidome dysregulation occurs, some findings being replicated in the plasma lipidome in a mouse OA model. One upregulated lipid is the sphingomyelin N-palmitoyl-D-erythro-sphingosylphosphorylcholine (d18:1/16:0), referred to here as SM. This study aimed to determine if SM causes joint pain and neuronal hyperexcitability in mice.
DESIGN: The effects of SM or a structurally related ceramide (CM) on mouse sensory neuron excitability were measured using patch-clamp electrophysiology, as well as the ability of intraarticular SM and CM to induce inflammatory pain in mice.
RESULTS: Incubation of sensory neurons with 1 µM SM decreased rheobase, compared to incubation with vehicle (p-adj = 0.0000146, 95% confidence interval (CI): 50.20, 76.73 pA) or CM (p-adj = 0.138, CI: 103.45, 171.55 pA). Similarly, SM induced mechanical hypersensitivity in mice compared to mice receiving vehicle (p-adj = 0.000003, 95% confidence interval (CI): 166.82, 251.63 g) or CM (p-adj = 0.055, 95% CI: 218.28, 268.12 g), which was coupled with a significant decrease in rheobase of knee-innervating neurons isolated from SM-injected mice compared to those receiving vehicle (p-adj = 0.0138, CI: 50.19, 76.73 pA) or CM (p-adj = 1.0, CI: 103.45, 171.55 pA).
CONCLUSIONS: The results generated demonstrate that a dysregulated lipidome can contribute to inflammatory OA pain, further work being necessary to determine the mechanism by which SM exerts its activity.
PMID:40691970 | DOI:10.1016/j.joca.2025.07.011
The SpyBLI cell-free pipeline for the rapid quantification of binding kinetics from crude samples
RSC Chem Biol. 2025 Jun 23. doi: 10.1039/d5cb00079c. Online ahead of print.
ABSTRACT
Accurate measurements of binding kinetics, encompassing equilibrium dissociation constant (K D), association rate (k on), and dissociation rate (k off), are critical for the development and optimisation of high-affinity binding proteins. However, such measurements require highly purified material and precise ligand immobilisation, limiting the number of binders that can be characterised within a reasonable timescale and budget. Here, we present the SpyBLI method, a rapid and cost-effective biolayer interferometry (BLI) pipeline that leverages the SpyCatcher003-SpyTag003 covalent association, eliminating the need for both binder purification and concentration determination. This approach allows for accurate binding-kinetic measurements to be performed directly from crude mammalian-cell supernatants or cell-free expression blends. We also introduce a linear gene fragment design that enables reliable expression in cell-free systems without any PCR or cloning steps, allowing binding kinetics data to be collected in under 24 hours from receiving inexpensive DNA fragments, with minimal hands-on time. We demonstrate the method's broad applicability using a range of nanobodies and single-chain antibody variable fragments (scFvs), with affinity values spanning six orders of magnitude. By minimising sample preparation and employing highly controlled, ordered sensor immobilisation, our workflow delivers reliable kinetic measurements from crude mixtures without sacrificing precision. We expect that the opportunity to carry out rapid and accurate binding measurements in good throughput should prove especially valuable for binder engineering, the screening of next-generation sequencing-derived libraries, and computational protein design, where large numbers of potential binders for the same target must be rapidly and accurately characterised to enable iterative refinement and candidate selection.
PMID:40655043 | PMC:PMC12247212 | DOI:10.1039/d5cb00079c
Clinical Implications of Pharmacogenomics of β<sub>1</sub>-Adrenoceptor for Anesthesia
J Cardiothorac Vasc Anesth. 2025 May 8:S1053-0770(25)00363-5. doi: 10.1053/j.jvca.2025.05.006. Online ahead of print.
ABSTRACT
Genetic polymorphisms within the β1-adrenoceptor are common within the population. Although not directly causative of disease, accumulating evidence supports that they have significant molecular and clinical effects, including altering the response to inotropes and β-blockers, as well as altering exercise capacity. Here, we summarize current evidence as relevant to anesthetists who treat patients with heart failure.
PMID:40581538 | DOI:10.1053/j.jvca.2025.05.006
Amyloid Forming Human Lysozyme Intermediates are Stabilized by Non-Native Amide-π Interactions
Adv Sci (Weinh). 2025 Jun 25:e03957. doi: 10.1002/advs.202503957. Online ahead of print.
ABSTRACT
Mutational variants of human lysozyme cause a rare but fatal hereditary systemic amyloidosis by populating an intermediate state that self-assembles into amyloid fibrils. However, this intermediate state is recalcitrant to detailed structural investigation, as it is only transiently and sparsely populated. Here, this work investigates the intermediate state of an amyloid-forming human lysozyme variant (I59T) using CEST and CPMG RD NMR at low pH. 15N CEST profiles probe the thermal unfolding of the native state into the denatured ensemble and reveal a distinct intermediate state. Global fitting of 15N CEST and CPMG data provides kinetic and thermodynamic parameters, characterizing the intermediate state populated at 0.6%. 1H CEST data further confirm the presence of the intermediate state displaying unusually high or low 1HN chemical shifts. To investigate the structural details of this intermediate state, this work uses molecular dynamics (MD) simulations, which recapitulate the experimentally observed folding pathway and free energy landscape. This work observes a high-energy intermediate state with a locally disordered β-domain and C-helix, stabilized by non-native hydrogen bonding and amide-π interactions, accounting for its anomalous 1H chemical shifts. Together, these NMR and MD data provide the first direct structural information on the intermediate state, offering insights into targeting lysozyme amyloidosis.
PMID:40557600 | DOI:10.1002/advs.202503957