Biology subjects

Radford, S. E.

Publications and source records attributed to Radford, S. E..

15 recordsLinked to original sources

bioRxiv2026

Distinct mechanistic pathways of early tauopathy revealed by MAPT mutations

Tau pathology underlies a broad spectrum of neurodegenerative disorders, collectively termed tauopathies, yet these diseases exhibit striking heterogeneity in their biological mechanisms and clinical outcomes. The basis for this heterogeneity remains poorly understood. Here, we address this question using knock-in mouse models expressing two distinct frontotemporal dementia-associated tau mutations to define how different tau variants drive divergent pathogenic programs in vivo. We find that the two mutations give rise to fundamentally different trajectories of tau pathogenesis. One trajectory is marked by progressive tau hyperphosphorylation and cytoskeletal destabilization occurring in the absence of detectable tau seed formation. In contrast, an alternative trajectory is characterized by tau hypophosphorylation, early seed formation, and alterations in nucleotide metabolism and chromatin organization, without overt cytoskeletal disruption. With aging, tau in this latter pathway transitions to a hyperphosphorylated state and forms mature fibrillar aggregates. Genetic enhancement of {beta}-amyloid selectively accelerates fibril formation, particularly in the model exhibiting early seeding. Together, these findings demonstrate that distinct tau mutations can engage separable pathogenic mechanisms, providing a biological framework for the heterogeneity observed across tauopathies, and highlighting the need for mechanism-informed therapeutic strategies and patient stratification. O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/705716v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@11eb652org.highwire.dtl.DTLVardef@153cc53org.highwire.dtl.DTLVardef@8b670eorg.highwire.dtl.DTLVardef@1afaad_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical abstractSchematic representation comparing two trajectories of tau pathology. The S305N mutation promotes a 4R isoform shift, cytoskeletal damage and synapse loss, and accumulation of soluble hyperphosphorylated tau. Tau remains soluble even at old ages. In contrast, the P301S mutation generates hypophosphorylated, seed-competent tau that forms fibrils. The effect of amyloid is slow in the S305N, but results in accelerated acceleration of pathology in the P301S. Figure made with Biorender.com.

neuroscience↗

bioRxiv2023

In situ cryo-electron tomography of beta-amyloid and tau in post-mortem Alzheimer's disease brain

A defining pathological feature of most neurodegenerative diseases is the assembly of proteins into amyloid that form disease-specific structures. In Alzheimers disease (AD) this is characterised by the deposition of amyloid-{beta} (A{beta}) and tau with AD-specific conformations. The in situ structure of amyloid in the human brain is unknown. Here, using cryogenic fluorescence microscopy (cryoFM)-targeted cryo-sectioning, cryo-focused ion beam scanning electron microscopy (cryoFIB-SEM) liftout and cryo-electron tomography (cryoET), we determined the in-tissue structure of {beta}-amyloid and tau pathology in fresh post-mortem AD donor brain. {beta}-amyloid plaques contained a mixture of fibrils and protofilaments arranged in parallel arrays and lattice-like structures, some of which were branched. Extracellular vesicles, extracellular droplets and open lipid bilayer sheets defined non-amyloid constituents of amyloid plaques. In contrast, tau inclusions were characterised by clusters of unbranched filaments. Subtomogram averaging of filaments within each cluster revealed distinct structures including variably twisted paired helical filaments (PHF) and chronic traumatic encephalopathy (CTE)-like tau filaments that were situated [~]1 m apart within two microscopic regions of pathology. Filaments within a cluster were similar to each other, but different between clusters, showing that fibril heterogeneity is spatially organised and influenced by the subcellular tissue environment. The in situ structural approaches outlined here for targeting specific proteins within human donor tissues have applications to a broad range of neurodegenerative diseases.

pathology↗

bioRxiv2023

Protein-lipid charge interactions control the folding of OMPs into asymmetric membranes

Biological membranes consist of two leaflets of phospholipid molecules that form a bilayer, and typically the composition of lipids in each leaflet is distinct. This asymmetry is created and maintained in vivo by dedicated biochemical pathways, but difficulties in creating stable asymmetric membranes in vitro have restricted our understanding of how bilayer asymmetry modulates the folding, stability and function of membrane proteins. Here we employ cyclodextrin mediated lipid exchange to generate asymmetric liposomes and use these to characterize the stability and folding kinetics of two bacterial outer membrane proteins (OMPs). We show that excess negative charge in the outer leaflet of a liposome impedes the membrane insertion and folding of OmpA and BamA, while excess negative charge in the inner leaflet accelerates their folding, relative to symmetric liposomes with the same membrane composition. Three positively charged residues in the extracellular loops of OmpA that play a critical role in folding are identified using molecular dynamics simulations and mutational analyses. Bioinformatic analysis was then used to identify a conserved patch of positive residues in the extracellular loops of OMPs generally that lies 6-8[A] from the membrane surface. Together, the the results rationalise the well known positive outside rule for OMP sequences and suggest new insights into the mechanisms that drive OMP folding and assembly in vitro and in vivo.

biophysics↗

bioRxiv2022

The in-tissue molecular architecture of β-amyloid pathology in the mammalian brain.

Amyloid plaques composed of extracellular focal deposition of A{beta} fibrils are a hallmark of Alzheimers disease (AD). Cryo-EM structures of A{beta} fibrils purified from human AD brain tissue post mortem have recently been determined. However, the molecular architecture of amyloid plaques in the context of fresh, unfixed mammalian brain tissue is unknown. Here, using cryogenic correlated light and electron tomography we report the native, in situ molecular architecture of A{beta} fibrils in the brain of a mouse model containing the Arctic familial AD mutation (AppNL-G-F) and an atomic model of Arctic A{beta} fibril purified from the brains of these animals. We show that in-tissue A{beta} fibrils are arranged in a lattice or in parallel bundles within a plaque, and are interdigitated by subcellular compartments, exosomes, extracellular droplets and extracellular multilamellar bodies. At the atomic level, the Arctic A{beta} fibril differs significantly from earlier structures of A{beta} amyloid extracted from AppNL-F mice models and human AD brain tissue, showing a striking effect of the Arctic mutation (E22G) on fibril structure. Cryo-electron tomography of ex vivo purified and in-tissue amyloid revealed an ensemble of additional fibrillar species, including thin protofilament-like rods and branched fibrils. Together, these results provide a structural model for the dense network architecture that characterises {beta}-amyloid plaque pathology.

neuroscience↗

bioRxiv2022

Tau amyloid polymorphism is shaped by local structural propensities of its protein sequence

Different tauopathies are characterized by specific amyloid filament folds that are conserved between patients. Disease-specific tau filament folds probably reflect the specific pathological contexts leading to their formation including isoforms or post-translational modifications. Little is known, however, as to whether and how intrinsic conformational tendencies of the tau sequence itself contribute to its polymorphism. Using cryo-EM structure determination we find that a short amyloidogenic C-terminal peptide consisting of residues 350-362 of the tau repeat domain adopts the same polymorphic conformations in isolation as it does in the context of major disease-associated protofilament folds. Biophysical characterisation and molecular modelling show that the amyloid conformations adopted by this peptide constitute core structural motifs stabilizing distinct disease-associated tau filament folds. In accordance this segment also contributes to the efficient propagation of human AD tau seeds in tau reporter cells while it is irrelevant to heparin-induced recombinant seeds. Our findings suggest that tau 350-362 is key to the propagation of disease-associated tau polymorphs and that the conformational preferences of this segment predispose to the topological diversity observed in tau filament folds.

biophysics↗

bioRxiv2021

Cooperative electrolyte-PEG interactions drive the signal amplification in a solid-state nanopore

Nanopore systems have emerged as a leading platform for the analysis of biomolecular complexes with single molecule resolution. However, the analysis of several analytes like short nucleic acids or proteins with nanopores represents a sensitivity challenge, because their translocation lead to small signals difficult to distinguish from the noise. Here, we report a simple method to enhance the signal to noise ratio in nanopore experiments by a simple modification of the solution used in nanopore sensing. The addition of poly-ethylene glycol (PEG) and the careful selection of the supporting electrolyte leads to large signal enhancement. We observed that the translocation dynamics are in good agreement with an established method that uses the lattice energy of an electrolyte to approximate the affinity of an ion to PEG. We identified CsBr as the optimal supporting electrolyte to complement PEG to enable the analysis of dsDNA at 500 kHz bandwidth, and the detection of dsDNA as short as 75 bp.

biophysics↗

bioRxiv2021

Sequence grammar underlying unfolding and phase separation of globular proteins

Aberrant phase separation of globular proteins is associated with many diseases. Here, we use a model protein system to understand how unfolded states of globular proteins drive phase separation and the formation of unfolded protein deposits (UPODs). For UPODs to form, the concentrations of unfolded molecules must be above a threshold value. Additionally, unfolded molecules must possess appropriate sequence grammars to drive phase separation. While UPODs recruit molecular chaperones, their compositional profiles are also influenced by synergistic physicochemical interactions governed by the sequence grammars of unfolded proteins and sequence features of cellular proteins. Overall, we find that the driving forces for phase separation and the compositional profiles of UPODs are governed by the sequence grammar of unfolded proteins. Our studies highlight the need for uncovering the sequence grammars of unfolded proteins that drive UPOD formation and lead to gain-of-function interactions whereby proteins are aberrantly recruited into UPODs. HighlightsO_LIUnfolded states of globular proteins phase separate to form UPODs in cells C_LIO_LIThe fraction of unfolded molecules and the sticker grammar govern phase separation C_LIO_LIHydrophobic residues act as stickers that engage in intermolecular interactions C_LIO_LISticker grammar also influences gain-of-function recruitment into aberrant UPODs C_LI

biophysics↗

bioRxiv2020

De novo design of transmembrane beta-barrels

The ability of naturally occurring transmembrane {beta}-barrel proteins (TMBs) to spontaneously insert into lipid bilayers and form stable transmembrane pores is a remarkable feat of protein evolution and has been exploited in biotechnology for applications ranging from single molecule DNA and protein sequencing to biomimetic filtration membranes. Because it has not been possible to design TMBs from first principles, these efforts have relied on re-engineering of naturally occurring TMBs that generally have a biological function very different from that desired. Here we leverage the power of de novo computational design coupled with a "hypothesis, design and test" approach to determine principles underlying TMB structure and folding, and find that, unlike almost all other classes of protein, locally destabilizing sequences in both the {beta}-turns and {beta}-strands facilitate TMB expression and global folding by modulating the kinetics of folding and the competition between soluble misfolding and proper folding into the lipid bilayer. We use these principles to design new eight stranded TMBs with sequences unrelated to any known TMB and show that they insert and fold into detergent micelles and synthetic lipid membranes. The designed proteins fold more rapidly and reversibly in lipid membranes than the TMB domain of the model native protein OmpA, and high resolution NMR and X-ray crystal structures of one of the designs are very close to the computational model. The ability to design TMBs from first principles opens the door to custom design of TMBs for biotechnology and demonstrates the value of de novo design to investigate basic protein folding problems that are otherwise hidden by evolutionary history. One sentence summarySuccess in de novo design of transmembrane {beta}-barrels reveals geometric and sequence constraints on the fold and paves the way to design of custom pores for sequencing and other single-molecule analytical applications.

synthetic biology↗

bioRxiv2020

Brazilin Removes Toxic alpha-Synuclein and Seeding Competent Assemblies from Parkinson Brain by Altering Conformational Equilibrium

Alpha-synuclein (-syn) fibrils, a major constituent of the neurotoxic Lewy Bodies in Parkinsons disease, form via nucleation dependent polymerization and can replicate by a seeding mechanism. Brazilin, a small molecule derived from red cedarwood trees in Brazil, has been shown to inhibit the fibrillogenesis of amyloid-beta (A{beta}) and -syn, prompting our inquiry in its mechanism of action. Here we test the effects of Brazilin on both seeded and unseeded -syn fibril formation and show that the natural polyphenol inhibits fibrillogenesis of -syn by a unique mechanism that is distinct from other polyphenols and is also distinct from its effect on A{beta}. Brazilin preserves the natively unfolded state of -syn by stabilizing the compact conformation of the -syn monomer over the aggregation-competent extended conformation. Molecular docking of Brazilin shows the molecule to interact both with unfolded -syn monomers and with the cross-{beta} sheet structure of -syn fibrils. Brazilin eliminates seeding competence of -syn assemblies from Parkinsons disease patient brain tissue, and treatment of pre-formed fibril assemblies with Brazilin significantly reduces their toxicity in primary neurons. Our findings suggest that Brazilin has substantial potential as a neuroprotective and therapeutic agent for Parkinsons Disease. Highlights- The natural polyphenol Brazilin binds to monomeric, oligomeric and fibrillar -syn - Brazilin shifts the equilibrium away from aggregation-competent monomer conformations - Brazilin inactivates seeding-competent -syn isolated from Parkinson patients brains - Brazilin detoxifies -syn aggregation intermediates and stabilizes mature amyloid fibrils Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=117 HEIGHT=200 SRC="FIGDIR/small/318220v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@447629org.highwire.dtl.DTLVardef@a9871dorg.highwire.dtl.DTLVardef@1e5a83dorg.highwire.dtl.DTLVardef@8843eb_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

PyXlinkViewer: a flexible tool for visualisation of protein chemical crosslinking data within the PyMOL molecular graphics system

Chemical crosslinking-mass spectrometry (XL-MS) is a valuable technique for gaining insights into protein structure and the organization of macromolecular complexes. XL-MS data yields inter-residue restraints that can be compared with high-resolution structural data. Distances greater than the crosslinker spacer-arm can reveal lowly-populated "excited" states of proteins/protein assemblies, or crosslinks can be used as restraints to generate structural models in the absence of structural data. Despite increasing uptake of XL-MS, there are few tools to enable rapid and facile mapping of XL-MS data onto high-resolution structures or structural models. PyXlinkViewer is a user-friendly plugin for PyMOL v2 that maps intra-protein, inter-protein and dead-end crosslinks onto protein structures/models and automates the calculation of inter-residue distances for the detected crosslinks. This enables rapid visualisation of XL-MS data, assessment of whether a set of detected crosslinks is congruent with structural data, and easy production of high-quality images for publication.

biochemistry↗

bioRxiv2019

Inter-domain dynamics in the chaperone SurA and multi-site binding to its unfolded outer membrane protein clients

The periplasmic chaperone SurA plays a key role in outer membrane protein (OMP) biogenesis. E. coli SurA comprises a core domain and two peptidylprolyl isomerase domains (P1 and P2), but how it binds its OMP clients and the mechanism(s) of its chaperone action remain unclear. Here, we have used chemical cross-linking, hydrogen-deuterium exchange, single-molecule FRET and molecular dynamics simulations to map the client binding site(s) on SurA and to interrogate the role of conformational dynamics of the chaperones domains in OMP recognition. We demonstrate that SurA samples a broad array of conformations in solution in which P2 primarily lies closer to the core/P1 domains than suggested by its crystal structure. Multiple binding sites for OMPs are located primarily in the core domain, with binding of the unfolded OMP resulting in conformational changes between the core/P1 domains. Together, the results portray a model in which unfolded OMP substrates bind in a cradle formed between the SurA domains, with structural flexibility between its domains assisting OMP recognition, binding and release.

biochemistry↗

bioRxiv2019

Structural insight into the formation of lipoprotein-β-barrel complexes by the β-barrel assembly machinery

The {beta}-barrel assembly machinery (BAM) inserts outer membrane {beta}-barrel proteins (OMPs) in the outer membrane of Gram-negative bacteria. In Enterobacteriacea, BAM also mediates export of the stress sensor lipoprotein RcsF to the cell surface by assembling RcsF-OMP complexes. Although BAM has been the focus of intense research due to its essential activity in generating and maintaining the outer membrane, how it functions remains poorly understood. Here, we report the crystal structure of the key BAM component BamA in complex with RcsF. BamA adopts an inward-open conformation, with the lateral gate to the membrane closed. The globular domain of RcsF is lodged deep inside the lumen of the BamA barrel, binding regions proposed to undergo an outward and lateral opening during OMP insertion. Our structural and biochemical data indicate a push-and-pull mechanism for RcsF export upon conformational cycling of BamA and provide a mechanistic explanation for how RcsF uses its interaction with BamA to detect envelope stress. Our data also suggest that the flux of incoming OMP substrates is involved in the control of BAM activity. Overall, the structural insights gleaned here elucidate a fundamental biological process and suggest a new avenue for antibiotic development.

microbiology↗

bioRxiv2019

Collagen I weakly interacts with the β-sheets of β2-microglobulin and enhances conformational exchange to induce amyloid formation

Amyloidogenesis is significant in both protein function and pathology. Amyloid formation of folded, globular proteins is commonly initiated by partial unfolding. However, how this unfolding event is triggered for proteins that are otherwise stable in their native environments is not well understood. The accumulation of the immunoglobulin protein {beta}2-microglobulin ({beta}2m) into amyloid plaques in the joints of long-term hemodialysis patients is the hallmark of Dialysis Related Amyloidosis (DRA). While {beta}2m does not form amyloid unassisted near neutral pH in vitro, the localization of {beta}2m deposits to joint spaces suggests a role for the local extracellular matrix (ECM) proteins, specifically collagens, in promoting amyloid formation. Indeed, collagen and other ECM components have been observed to facilitate {beta}2m amyloid formation, but the large size and anisotropy of the complex, combined with the low affinity of these interactions, has limited atomic-level elucidation of the amyloid-promoting mechanism by these molecules. Using solution NMR approaches that uniquely probe weak interactions and large complexes, we are able to derive binding interfaces for collagen I on {beta}2m and detect collagen I-induced {micro}s-ms timescale dynamics in the {beta}2m backbone. By combining solution NMR relaxation methods and 15N-dark state exchange saturation transfer experiments, we propose a model in which weak, multimodal collagen I-{beta}2m interactions promote exchange with a minor population of an amyloid-competent species to induce fibrillogenesis. The results portray the intimate role of the environment in switching an innocuous protein into an amyloid-competent state, rationalizing the localization of amyloid deposits in DRA.

biophysics↗

bioRxiv2019

Energy landscape steering in SecYEG mediates dynamic coupling in ATP driven protein translocation

The Sec translocon is a highly conserved membrane complex for transport of polypeptides across, or into, lipid bilayers. In bacteria, the core protein-channel SecYEG resides in the inner-membrane, through which secretion is powered by the cytosolic ATPase SecA. Here, we use single-molecule fluorescence to interrogate the dynamic state of SecYEG throughout the hydrolytic cycle of SecA. We show that the SecYEG channel fluctuates between open and closed states faster ([~]20-fold during transport) than ATP turnover; while the nucleotide status of SecA modulates the rates of opening and closure. Interestingly, a SecY variant (PrlA4), exhibiting faster protein transport, but unaffected ATPase rates, increases the dwell time in the open state, facilitating pre-protein diffusion through the pore; thereby improving the efficiency of translocation. Thus, contrary to prevailing structure-based models, SecYEG plays an integral part in the translocation mechanism through dynamic allosteric coupling in which SecA steers the energy landscape of the protein-channel.

biophysics↗

bioRxiv2018

Dynamic action of the Sec machinery during initiation, protein translocation and termination revealed by single molecule fluorescence

Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. This is mediated, for the majority of proteins, by the highly conserved Sec machinery. The bacterial translocon - SecYMKEG - resides in the plasma membrane, where translocation is driven through rounds of ATP hydrolysis by the cytoplasmic SecA ATPase, and the proton motive force (PMF). We have used single molecule Forster resonance energy transfer (FRET) alongside a combination of confocal and total internal reflection microscopy to gain access to SecY pore dynamics and translocation kinetics on timescales spanning milliseconds to minutes. This allows us to dissect and characterise the translocation process in unprecedented detail. We show that SecA, signal sequence, pre-protein and ATP hydrolysis each have important and specific roles in unlocking and opening the Sec channel, priming it for transport. After channel opening, translocation proceeds in two phases: an initiation phase independent of substrate length, and a length-dependent transport phase with an intrinsic translocation rate of ~ 40 amino acids per second for the model pre-protein substrate proOmpA. The initiation and translocation phases are both coupled to ATP hydrolysis while termination is ATP-independent. Distributions of translocation rates reflect the stochastic nature of the translocation process and are consistent with the recently proposed Brownian ratchet model [Allen et al. doi: 10.7554/eLife.15598]. The results allow us unparalleled access to the kinetics of the complex reaction and provide a framework for understanding the molecular mechanism of protein secretion.

biophysics↗