ORCID Profile
0000-0002-3079-8039
Current Organisations
University of Leeds
,
The Royal Society / University of Leeds
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Publisher: SAGE Publications
Date: 04-2009
DOI: 10.1255/EJMS.947
Abstract: Detailed knowledge of the tertiary and quaternary structure of proteins and protein complexes is of immense importance in understanding their functionality. Similarly, variations in the conformational states of proteins form the underlying mechanisms behind many biomolecular processes, numerous of which are disease-related. Thus, the availability of reliable and accurate biophysical techniques that can provide detailed information concerning these issues is of paramount importance. Ion mobility spectrometry (IMS) coupled to mass spectrometry (MS) offers a unique opportunity to separate multi-component biomolecular entities and to measure the molecular mass and collision cross-section of in idual components in a single, rapid (≤ 2 min) experiment, providing 3D-architectural information directly. Here we report a method of calibrating a commercially available electrospray ionisation (ESI)-travelling wave ion mobility spectrometry (TWIMS)–mass spectrometer using known cross-sectional areas determined for a range of biomolecules by conventional IMS-MS. Using this method of calibration, we have analysed a range of proteins of differing mass and 3D architecture in their native conformations by ESI-TWIMS-MS and found that the cross-sectional areas measured in this way compare extremely favourably with cross-sectional areas calculated using an in-house computing method based on Protein Data Bank NMR-derived co-ordinates. This not only provides a high degree of confidence in the calibration method, but also suggests that the gas phase ESI-TWIMS-MS measurements relate well to solution-based measurements derived from other biophysical techniques. In order to determine which instrumental parameters affect the ESI-TWIMS-MS cross-sectional area calibration, a systematic study of the parameters used to optimise TWIMS drift time separations has been carried out, observing the effect each parameter has on drift times and IMS resolution. Finally, the ESI-TWIMS-MS cross-sectional area calibration has been applied to the analysis of the amyloidogenic protein β 2 -microglobulin and measurements for three co-populated conformational families, present under denaturing conditions, have been made: the folded, partially unfolded and unfolded states.
Publisher: American Chemical Society (ACS)
Date: 09-08-2017
Publisher: Proceedings of the National Academy of Sciences
Date: 15-07-2002
Abstract: Dissociation of human β-2-microglobulin (β 2 m) from the heavy chain of the class I HLA complex is a critical first step in the formation of amyloid fibrils from this protein. As a consequence of renal failure, the concentration of circulating monomeric β 2 m increases, ultimately leading to deposition of the protein into amyloid fibrils and development of the disorder, dialysis-related amyloidosis. Here we present the crystal structure of a monomeric form of human β 2 m determined at 1.8-Å resolution that reveals remarkable structural changes relative to the HLA-bound protein. These involve the restructuring of a β bulge that separates two short β strands to form a new six-residue β strand at one edge of this β sandwich protein. These structural changes remove key features proposed to have evolved to protect β sheet proteins from aggregation [Richardson, J. & Richardson, D. (2002) Proc. Natl. Acad. Sci. USA 99, 2754–2759] and replaces them with an aggregation-competent surface. In combination with solution studies using 1 H NMR, we show that the crystal structure presented here represents a rare species in solution that could provide important clues about the mechanism of amyloid formation from the normally highly soluble native protein.
Publisher: Springer Science and Business Media LLC
Date: 25-07-2016
DOI: 10.1038/NSMB.3266
Publisher: Elsevier BV
Date: 09-2011
Publisher: Royal Society of Chemistry (RSC)
Date: 2008
DOI: 10.1039/B813504E
Abstract: The separative and analytical power of ion mobility spectrometry-mass spectrometry combined with photo-induced cross-linking of site-specifically incorporated trifluoromethyldiazirine provides a powerful approach towards structural characterisation of amyloid fibrils.
Publisher: eLife Sciences Publications, Ltd
Date: 17-07-2019
Publisher: American Chemical Society (ACS)
Date: 07-2000
DOI: 10.1021/BI000276J
Abstract: Dialysis-related amyloidosis (DRA) involves the aggregation of beta(2)-microglobulin (beta(2)m) into amyloid fibrils. Using Congo red and thioflavin-T binding, electron microscopy, and X-ray fiber diffraction, we have determined conditions under which recombinant monomeric beta(2)m spontaneously associates to form fibrils in vitro. Fibrillogenesis is critically dependent on the pH and the ionic strength of the solution, with low pH and high ionic strength favoring fibril formation. The morphology of the fibrils formed varies with the growth conditions. At pH 4 in 0.4 M NaCl the fibrils are approximately 10 nm wide, relatively short (50-200 nm), and curvilinear. By contrast, at pH 1.6 the fibrils formed have the same width and morphology as those formed at pH 4 but extend to more than 600 nm in length. The dependence of fibril growth on ionic strength has allowed the conformational properties of monomeric beta(2)m to be determined under conditions where fibril growth is impaired. Circular dichroism studies show that titration of one or more residues with a pK(a) of 4.7 destabilizes native beta(2)m and generates a partially unfolded species. On average, these molecules retain significant secondary structure and have residual, non-native tertiary structure. They also bind the hydrophobic dye 1-anilinonaphthalene-8-sulfonic acid (ANS), show line broadening in one-dimensional (1)H NMR spectra, and are weakly protected from hydrogen exchange. Further acidification destabilizes this species, generating a second, more highly denatured state that is less fibrillogenic. These data are consistent with a model for beta(2)m fibrillogenesis in vitro involving the association of partially unfolded molecules into ordered fibrillar assemblies.
Publisher: Elsevier BV
Date: 10-2001
Publisher: Springer Science and Business Media LLC
Date: 05-2020
DOI: 10.1038/S41467-020-15702-1
Abstract: 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 its mechanisms of client binding and chaperone function have remained unclear. Here, we use chemical cross-linking, hydrogen-deuterium exchange mass spectrometry, single-molecule FRET and molecular dynamics simulations to map the client binding site(s) on SurA and interrogate the role of conformational dynamics in OMP recognition. We demonstrate that SurA s les an array of conformations in solution in which P2 primarily lies closer to the core/P1 domains than suggested in the SurA crystal structure. OMP binding sites are located primarily in the core domain, and OMP binding results in conformational changes between the core/P1 domains. Together, the results suggest that unfolded OMP substrates bind in a cradle formed between the SurA domains, with structural flexibility between domains assisting OMP recognition, binding and release.
Publisher: Springer Science and Business Media LLC
Date: 14-12-2020
DOI: 10.1038/S42003-020-01419-W
Abstract: The β-barrel assembly machinery (BAM) catalyses the folding and insertion of β-barrel outer membrane proteins (OMPs) into the outer membranes of Gram-negative bacteria by mechanisms that remain unclear. Here, we present an ensemble of cryoEM structures of the E. coli BamABCDE (BAM) complex in lipid nanodiscs, determined using multi-body refinement techniques. These structures, supported by single-molecule FRET measurements, describe a range of motions in the BAM complex, mostly localised within the periplasmic region of the major subunit BamA. The β-barrel domain of BamA is in a ‘lateral open’ conformation in all of the determined structures, suggesting that this is the most energetically favourable species in this bilayer. Strikingly, the BAM-containing lipid nanodisc is deformed, especially around BAM’s lateral gate. This distortion is also captured in molecular dynamics simulations, and provides direct structural evidence for the lipid ‘disruptase’ activity of BAM, suggested to be an important part of its functional mechanism.
Publisher: Elsevier BV
Date: 11-2015
Publisher: Springer Science and Business Media LLC
Date: 07-07-2021
DOI: 10.1038/S41467-021-24432-X
Abstract: The folding of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the β-barrel assembly machinery (BAM). How lateral opening in the β-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.
Publisher: Cold Spring Harbor Laboratory
Date: 17-06-2020
DOI: 10.1101/2020.06.16.154773
Abstract: 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 rotein 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.
Publisher: Elsevier BV
Date: 07-2003
DOI: 10.1016/S0022-2836(03)00688-0
Abstract: Beta 2-microglobulin (beta(2)m) is known to form amyloid fibrils de novo in vitro under acidic conditions (below pH 4.8). Fibril formation at neutral pH, however, has only been observed by deletion of the N-terminal six residues by the addition of pre-assembled seeds or in the presence of Cu(2+). Based on these observations, and other structural data, models for fibril formation of beta(2)m have been proposed that involve the fraying of the N and C-terminal beta-strands and the consequent loss of edge strand protective features. Here, we examine the role of the N and C-terminal strands in the initiation of fibrillogenesis of beta(2)m by creating point mutations in strands A and G and comparing the properties of the resulting proteins with variants containing similar mutations elsewhere in the protein. We show that truncation of buried hydrophobic side-chains in strands A and G promotes rapid fibril formation at neutral pH, even in unseeded reactions, and increases the rate of fibril formation under acidic conditions. By contrast, similar mutations created in the remaining seven beta-strands of the native protein have little effect on the rate or pH dependence of fibril formation. The data are consistent with the view that perturbation of the N and C-terminal edge strands is an important feature in the generation of assembly-competent states of beta(2)m.
Publisher: Wiley
Date: 21-11-2018
Publisher: Elsevier BV
Date: 07-2003
DOI: 10.1016/S0022-2836(03)00687-9
Abstract: Beta-2-microglobulin (beta(2)m) has been shown to form amyloid fibrils with distinct morphologies under acidic conditions in vitro. Short, curved fibrils (<600 nm in length), form rapidly without a lag phase, with a maximum rate at pH 3.5. By contrast, fibrils with a long (approximately 1 microm), straight morphology are produced by incubation of the protein at pH< or =3.0. Both fibril types display Congo red birefringence, bind Thioflavin-T and have X-ray fibre diffraction patterns consistent with a cross-beta structure. In order to investigate the role of different partially folded states in generating fibrils of each type, and to probe the effect of protein stability on amyloid formation, we have undertaken a detailed mutagenesis study of beta(2)m. Thirteen variants containing point mutations in different regions of the native protein were created and their structure, stability and fibril forming propensities were investigated as a function of pH. By altering the stability of the native protein in this manner, we show that whilst destabilisation of the native state is important in the generation of amyloid fibrils, population of specific denatured states is a pre-requisite for amyloid formation from this protein. Moreover, we demonstrate that the formation of fibrils with different morphologies in vitro correlates with the relative population of different precursor states.
Publisher: Wiley
Date: 09-2001
DOI: 10.1110/PS.4901
Abstract: The aggregation of beta(2)-microglobulin (beta(2)m) into amyloid fibrils occurs in the condition known as dialysis-related amyloidosis (DRA). The protein has a beta-sandwich fold typical of the immunoglobulin family, which is stabilized by a highly conserved disulphide bond linking Cys25 and Cys80. Oxidized beta(2)m forms amyloid fibrils rapidly in vitro at acidic pH and high ionic strength. Here we investigate the role of the single disulphide bond of beta(2)m in amyloidosis in vitro. We show that reduction of the disulphide bond destabilizes the native protein such that non-native molecules are populated at neutral pH. These species are prone to oligomerization but do not form amyloid fibrils when incubated for up to 8 mo at pH 7.0 in 0.4 M NaCl. Over the pH range 4.0-1.5 in the presence of 0.4 M NaCl, however, amyloid fibrils of reduced beta(2)m are formed. These fibrils are approximately 10 nm wide, but are shorter and assemble more rapidly than those produced from the oxidized protein. These data show that population of non-native conformers of beta(2)m at neutral pH by reduction of its single disulphide bond is not sufficient for amyloid formation. Instead, association of one or more specific partially unfolded molecules formed at acid pH are necessary for the formation of beta(2)m amyloid in vitro. Further experiments will now be needed to determine the role of different oligomeric species of beta(2)m in the toxicity of the protein in vivo.
Publisher: Proceedings of the National Academy of Sciences
Date: 29-03-2010
Abstract: The key to understanding amyloid disease is the characterization of oligomeric species formed during the early stages of fibril assembly. Here we have used electrospray ionisation-ion mobility spectrometry-mass spectrometry to identify and structurally characterize the oligomers formed during amyloid assembly from β 2 -microglobulin ( β 2 m). β 2 m oligomers are shown to have collision cross-sections consistent with monomeric units arranged in elongated assemblies prior to fibril formation. Direct observation, separation, and quantification of transient oligomeric species reveals that monomers to tetramers are populated through the lag phase with no evidence for the significant population of larger oligomeric species under the conditions employed. The dynamics of each oligomeric species were monitored directly within the ensemble at concentrations commensurate with amyloid formation by observing the subunit exchange of 14 N- and 15 N- labeled oligomers. Analysis of the data revealed a decrease in oligomer dynamics concomitant with increasing oligomer size and the copopulation of dynamic dimeric and trimeric species with more stable trimeric and tetrameric species. The results presented map the events occurring during the lag phase of fibril formation and give a clear insight into the structural characteristics and dynamic nature of the β 2 m oligomers, demonstrating the existence of elongated assemblies arising from an intact amyloidogenic protein during fibril formation.
Publisher: Elsevier BV
Date: 07-2003
DOI: 10.1016/S0022-2836(03)00583-7
Abstract: The kinetics of spontaneous assembly of amyloid fibrils of wild-type beta(2)-microglobulin (beta(2)M) in vitro, under acid conditions (pH 2.5) and low ionic strength, has been followed using thioflavin-T (ThT) binding. In parallel experiments, the morphology of the different fibrillar species present at different time-points during the growth process were characterised using tapping-mode atomic force microscopy (TM-AFM) in air and negative stain electron microscopy (EM). The thioflavin-T assay shows a characteristic lag phase during which the nucleation of fibrils occurs before a rapid growth in fibril density. The volume of fibrils deposited on mica measured from TM-AFM images at each time-point correlates well with the fluorescence data. TM-AFM and negative-stain EM revealed the presence of various kinds of protein aggregates in the lag phase that disappear concomitantly with a rise in the density of amyloid fibrils, suggesting that these aggregates precede fibril growth and may act as nucleation sites. Three distinct morphologies of mature amyloid fibrils were observed within a single growth experiment, as observed previously for the wild-type protein and the variant N17D. Additional supercoiled morphologies of the lower-order fibrils were observed. Comparative height analysis from the TM-AFM data allows each of the mature fibril types and single protofilaments to be identified unambiguously, and reveals that the assembly occurs via a hierarchy of morphological states.
Publisher: Wiley
Date: 03-07-2020
DOI: 10.1002/PRO.3902
Publisher: Wiley
Date: 14-11-2012
Publisher: Springer Science and Business Media LLC
Date: 08-1990
DOI: 10.1038/NBT0890-741
Abstract: We transformed Aspergillus niger with the full length cDNA gene encoding hen egg-white lysozyme (HEWL) and its secretion signal sequence. Lysozyme levels up to 12 mg/l were secreted when expression was controlled by the A. awamori glucoamylase (GAM) promoter and 1 mg/l when controlled by the A. nidulans glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter. N-terminal sequence analysis of the recombinant protein indicated that the signal peptide was correctly processed by the A. niger secretory apparatus. The specific catalytic activity of the recombinant protein was identical to that of authentic hen lysozyme. The recombinant HEWL was examined by 2D 1H-NMR spectroscopy and shown to have a spectrum identical to that of authentic HEWL indicating that the protein was correctly folded.
Publisher: Elsevier BV
Date: 02-2011
DOI: 10.1016/J.JMB.2010.12.006
Abstract: The self-assembly of specific proteins to form insoluble amyloid fibrils is a characteristic feature of a number of age-related and debilitating diseases. Lipid-free human apolipoprotein C-II (apoC-II) forms characteristic amyloid fibrils and is one of several apolipoproteins that accumulate in amyloid deposits located within atherosclerotic plaques. X-ray diffraction analysis of aligned apoC-II fibrils indicated a simple cross-β-structure composed of two parallel β-sheets. Examination of apoC-II fibrils using transmission electron microscopy, scanning transmission electron microscopy, and atomic force microscopy indicated that the fibrils are flat ribbons composed of one apoC-II molecule per 4.7-Å rise of the cross-β-structure. Cross-linking results using single-cysteine substitution mutants are consistent with a parallel in-register structural model for apoC-II fibrils. Fluorescence resonance energy transfer analysis of apoC-II fibrils labeled with specific fluorophores provided distance constraints for selected donor-acceptor pairs located within the fibrils. These findings were used to develop a simple 'letter-G-like' β-strand-loop-β-strand model for apoC-II fibrils. Fully solvated all-atom molecular dynamics (MD) simulations showed that the model contained a stable cross-β-core with a flexible connecting loop devoid of persistent secondary structure. The time course of the MD simulations revealed that charge clusters in the fibril rearrange to minimize the effects of same-charge interactions inherent in parallel in-register models. Our structural model for apoC-II fibrils suggests that apoC-II monomers fold and self-assemble to form a stable cross-β-scaffold containing relatively unstructured connecting loops.
Publisher: Springer Science and Business Media LLC
Date: 30-09-2016
DOI: 10.1038/NCOMMS12865
Abstract: The β-barrel assembly machinery (BAM) is a ∼203 kDa complex of five proteins (BamA–E), which is essential for viability in E. coli . BAM promotes the folding and insertion of β-barrel proteins into the outer membrane via a poorly understood mechanism. Several current models suggest that BAM functions through a ‘lateral gating’ motion of the β-barrel of BamA. Here we present a cryo-EM structure of the BamABCDE complex, at 4.9 Å resolution. The structure is in a laterally open conformation showing that gating is independent of BamB binding. We describe conformational changes throughout the complex and interactions between BamA, B, D and E, and the detergent micelle that suggest communication between BAM and the lipid bilayer. Finally, using an enhanced reconstitution protocol and functional assays, we show that for the outer membrane protein OmpT, efficient folding in vitro requires lateral gating in BAM.
Publisher: eLife Sciences Publications, Ltd
Date: 25-09-2019
DOI: 10.7554/ELIFE.46574
Abstract: Transient oligomers are commonly formed in the early stages of amyloid assembly. Determining the structure(s) of these species and defining their role(s) in assembly is key to devising new routes to control disease. Here, using a combination of chemical kinetics, NMR spectroscopy and other biophysical methods, we identify and structurally characterize the oligomers required for amyloid assembly of the protein ΔN6, a truncation variant of human β2-microglobulin (β2m) found in amyloid deposits in the joints of patients with dialysis-related amyloidosis. The results reveal an assembly pathway which is initiated by the formation of head-to-head non-toxic dimers and hexamers en route to amyloid fibrils. Comparison with inhibitory dimers shows that precise subunit organization determines amyloid assembly, while dynamics in the C-terminal strand hint to the initiation of cross-β structure formation. The results provide a detailed structural view of early amyloid assembly involving structured species that are not cytotoxic.
Publisher: Elsevier BV
Date: 11-2017
Publisher: Springer Science and Business Media LLC
Date: 08-06-2022
DOI: 10.1038/S42003-022-03502-W
Abstract: Correct folding of outer membrane proteins (OMPs) into the outer membrane of Gram-negative bacteria depends on delivery of unfolded OMPs to the β-barrel assembly machinery (BAM). How unfolded substrates are presented to BAM remains elusive, but the major OMP chaperone SurA is proposed to play a key role. Here, we have used hydrogen deuterium exchange mass spectrometry (HDX-MS), crosslinking, in vitro folding and binding assays and computational modelling to show that the core domain of SurA and one of its two PPIase domains are key to the SurA-BAM interaction and are required for maximal catalysis of OMP folding. We reveal that binding causes changes in BAM and SurA conformation and/or dynamics distal to the sites of binding, including at the BamA β1-β16 seam. We propose a model for OMP biogenesis in which SurA plays a crucial role in OMP delivery and primes BAM to accept substrates for folding.
Publisher: American Chemical Society (ACS)
Date: 12-2007
Publisher: Elsevier BV
Date: 09-2018
DOI: 10.1016/J.YMETH.2018.02.020
Abstract: The last ∼25 years has seen mass spectrometry (MS) emerge as an integral method in the structural biology toolkit. In particular, MS has enabled the structural characterization of proteins and protein assemblies that have been intractable by other methods, especially those that are large, heterogeneous or transient, providing experimental evidence for their structural organization in support of, and in advance of, high resolution methods. The most recent frontier conquered in the field of MS-based structural biology has been the application of established methods for studying water soluble proteins to the more challenging targets of integral membrane proteins. The power of MS in obtaining structural information has been enabled by advances in instrumentation and the development of hyphenated mass spectrometry-based methods, such as ion mobility spectrometry-MS, chemical crosslinking-MS and other chemical labelling/footprinting-MS methods. In this review we detail the insights garnered into the structural biology of membrane proteins by applying such techniques. Application and refinement of these methods has yielded unprecedented insights in many areas, including membrane protein conformation, dynamics, lipid/ligand binding, and conformational perturbations due to ligand binding, which can be challenging to study using other methods.
Publisher: Springer Science and Business Media LLC
Date: 02-1997
DOI: 10.1038/385787A0
Abstract: Tissue deposition of soluble proteins as amyloid fibrils underlies a range of fatal diseases. The two naturally occurring human lysozyme variants are both amyloidogenic, and are shown here to be unstable. They aggregate to form amyloid fibrils with transformation of the mainly helical native fold, observed in crystal structures, to the amyloid fibril cross-beta fold. Biophysical studies suggest that partly folded intermediates are involved in fibrillogenesis, and this may be relevant to amyloidosis generally.
Publisher: Springer Science and Business Media LLC
Date: 15-07-2022
DOI: 10.1038/S41467-022-31767-6
Abstract: ATP-independent chaperones like trigger factor are generally assumed to play passive roles in protein folding by acting as holding chaperones. Here we show that trigger factor plays a more active role. Consistent with a role as an aggregation inhibiting chaperone, we find that trigger factor rapidly binds to partially folded glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and prevents it from non-productive self-association by shielding oligomeric interfaces. In the traditional view of holding chaperone action, trigger factor would then be expected to transfer its client to a chaperone foldase system for complete folding. Unexpectedly, we noticed that GAPDH folds into a monomeric but otherwise rather native-like intermediate state while trigger factor-bound. Upon release from trigger factor, the mostly folded monomeric GAPDH rapidly self-associates into its native tetramer and acquires enzymatic activity without needing additional folding factors. The mechanism we propose here for trigger factor bridges the holding and folding activities of chaperone function.
Publisher: American Chemical Society (ACS)
Date: 08-05-2017
Publisher: Wiley
Date: 21-11-2018
Publisher: Cold Spring Harbor Laboratory
Date: 20-08-2021
DOI: 10.1101/2021.08.20.457073
Abstract: 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. Unfolded states of globular proteins phase separate to form UPODs in cells The fraction of unfolded molecules and the sticker grammar govern phase separation Hydrophobic residues act as stickers that engage in intermolecular interactions Sticker grammar also influences gain-of-function recruitment into aberrant UPODs
Publisher: Cold Spring Harbor Laboratory
Date: 10-02-2023
DOI: 10.1101/2023.02.10.527650
Abstract: Oligomeric species populated during α-synuclein aggregation are considered key drivers of neurodegeneration in Parkinson’s disease. However, their structure and the molecular determinants driving their conversion to fibrils remain elusive. In this work, we determined the symmetry and architecture of α-synuclein oligomers, dissecting the conformational properties of in idual chains within these toxic assemblies. We demonstrate that the NAC domain is insufficient to promote oligomer to fibril conversion instead, this transition is controlled by a short α-synuclein N-terminal motif. A missense mutation causing early-onset Parkinson’s disease remodels this N-terminal region conformation, which results in a population of long-lived oligomers less susceptible to disaggregation by the human Hsp70 machinery. Our results provide a structural understanding of oligomer to amyloid conversion and identify targets for therapeutic intervention. α-Synuclein oligomers are symmetric and well-organized particles with a short N-terminal region controlling fibril conversion.
Publisher: Springer Science and Business Media LLC
Date: 22-06-2020
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
Start Date: 2008
End Date: 2009
Funder: Biotechnology and Biological Sciences Research Council
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