Sheena E Radford

Sheena E Radford
University of Leeds

Doctor of Philosophy

About

440
Publications
48,752
Reads
How we measure 'reads'
A 'read' is counted each time someone views a publication summary (such as the title, abstract, and list of authors), clicks on a figure, or views or downloads the full-text. Learn more
26,508
Citations

Publications

Publications (440)
Article
How do bacteria assemble transmembrane β-barrels into their outer membrane in the absence of an energy source? New structures and experiments from Doyle et al. suggest that the β-barrel assembly machinery (BAM) co-opts the power of membrane elastic tension to help complete the folding of β-barrel outer membrane proteins.
Article
Full-text available
Human islet amyloid polypeptide (hIAPP) self-assembles into amyloid fibrils which deposit in pancreatic islets of type 2 diabetes (T2D) patients. Here, we applied chemical kinetics to study the mechanism of amyloid assembly of wild-type hIAPP and its more amyloidogenic natural variant S20G. We show that the aggregation of both peptides involves pri...
Preprint
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. H...
Article
Full-text available
Aggregation of β2 microglobulin (β2m) into amyloid fibrils is associated with systemic amyloidosis, caused by the deposition of amyloid fibrils containing the wild-type protein and its truncated variant, ΔN6 β2m, in haemo-dialysed patients. A second form of familial systemic amyloidosis caused by the β2m variant, D76N, results in amyloid deposits i...
Article
Full-text available
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 fr...
Article
Although critically important for protein function, post-translational modifications are complex and notoriously difficult to study. Now, the effects of O-GlcNAcylation on chaperone activity and the accompanying inhibition of amyloid fibril formation have been revealed, potentially yielding new routes to combat neurodegeneration.
Article
Transmembrane β-barrel proteins (TMBs) are of great interest for single-molecule analytical technologies because they can spontaneously fold and insert into membranes and form stable pores, but the range of pore properties that can be achieved by repurposing natural TMBs is limited. We leverage the power of de novo computational design coupled with...
Article
Full-text available
NMR studies and X-ray crystallography have shown that the structures of the 99-residue amyloidogenic protein β2-microglobulin (β2m) and its more aggregation-prone variant, D76N, are indistinguishable, and hence, the reason for the striking difference in their aggregation propensities remains elusive. Here, we have employed two protein footprinting...
Chapter
The thermodynamic stabilities of membrane proteins are of fundamental interest to provide a biophysical description of their structure-function relationships because energy determines conformational populations. In addition, structure-energy relationships can be exploited in membrane protein design and in synthetic biology. To determine the thermod...
Article
Alpha-synuclein (α-syn) fibrils, a major constituent of the neurotoxic Lewy Bodies in Parkinson’s 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 (Aβ) and α-syn as well...
Preprint
Full-text available
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 fr...
Article
Full-text available
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...
Article
Full-text available
Protein–protein interactions (PPIs) are involved in many of life’s essential biological functions yet are also an underlying cause of several human diseases, including amyloidosis. The modulation of PPIs presents opportunities to gain mechanistic insights into amyloid assembly, particularly through the use of methods which can trap specific interme...
Article
Full-text available
Amyloid plaques are a pathological hallmark of Alzheimer’s disease. The major component of these plaques are highly ordered amyloid fibrils formed by amyloid-β (Aβ) peptides. However, whilst Aβ amyloid fibril assembly has been subjected to detailed and extensive analysis in vitro, these studies may not reproduce how Aβ fibrils assemble in the brain...
Article
Full-text available
Aggregation of the peptide hormone amylin into amyloid deposits is a pathological hallmark of type-2 diabetes (T2D). While no causal link between T2D and amyloid has been established, the S20G mutation in amylin is associated with early-onset T2D. Here we report cryo-EM structures of amyloid fibrils of wild-type human amylin and its S20G variant. T...
Article
Full-text available
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Article
Full-text available
Oligomers which form during amyloid fibril assembly are considered to be key contributors towards amyloid disease. However, understanding how such intermediates form, their structure, and mechanisms of toxicity presents significant challenges due to their transient and heterogeneous nature. Here, we discuss two different strategies for addressing t...
Preprint
Full-text available
The ability of naturally occurring transmembrane β-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. Becau...
Article
Full-text available
Determining the structure of the (oligomeric) intermediates that form during the self‐assembly of amyloidogenic peptides is challenging because of their heterogeneous and dynamic nature. Thus, there is need for methodology to analyze the underlying molecular structure of these transient species. In this work, a combination of fluorescence quenching...
Article
Studies in experimental animals show transmissibility of amyloidogenic proteins associated with prion diseases, Alzheimer's disease, Parkinson's disease, and other neurodegenerative diseases. Although these data raise potential concerns for public health, convincing evidence for human iatrogenic transmission only exists for prions and amyloid β aft...
Preprint
Full-text available
Alpha-synuclein (α-syn) fibrils, a major constituent of the neurotoxic Lewy Bodies in Parkinson’s 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 (Aβ) and α-syn, prompti...
Preprint
p> Protein-protein interactions (PPIs) are involved in many of life’s essential biological functions yet are also an underlying cause of several human diseases, including amyloidosis. The modulation of PPIs presents opportunities to gain mechanistic insights into amyloid assembly, particularly through the use of methods which can trap specific int...
Article
Full-text available
The β-barrel assembly machinery (BAM) inserts outer membrane β-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. Here, we report the crystal structure of the key BAM component BamA in compl...
Article
Full-text available
The D76N variant of human β2-microglobulin (β2m) is the causative agent of a hereditary amyloid disease. Interestingly, D76N-associated amyloidosis has a distinctive pathology compared with aggregation of wild-type (WT) β2m which occurs in dialysis-related amyloidosis. A folding intermediate of WT-β2m, known as the IT-state, which contains a non-na...
Article
Full-text available
Nanopore analysis of nucleic acid is now routine, but detection of proteins remains challenging. Here, we report the systematic characterization of the effect of macromolecular crowding on the detection sensitivity of a solid-state nanopore for circular and linearized DNA plasmids, globular proteins (β-galactosidase), and filamentous proteins (α-sy...
Article
Full-text available
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 “exci...
Preprint
Full-text available
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 'exci...
Article
Full-text available
β-barrel outer membrane proteins (OMPs) represent the major proteinaceous component of the outer membrane (OM) of Gram-negative bacteria. These proteins perform key roles in cell structure and morphology, nutrient acquisition, colonisation and invasion, and protection against external toxic threats such as antibiotics. To become functional, OMPs mu...
Article
Full-text available
The development of therapeutic monoclonal antibodies (mAbs) can be hindered by their tendency to aggregate throughout their lifetime, which can illicit immunogenic responses and render mAb manufacturing unfeasible. Consequently, there is a need to identify mAbs with desirable thermodynamic stability, solubility, and lack of self‐association. These...
Article
Full-text available
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,...
Article
In a recent study, Hervas et al. extracted Orb2 fibrils, that are involved in long-term memory formation, from Drosophila brains, characterised their function, and determined their structure using cryo-electron microscopy (cryo-EM). The fibrils show a remarkable resemblance to amyloid β (Aβ) fibrils associated with Alzheimer’s disease, highlighting...
Article
Full-text available
Protein biopharmaceuticals are highly successful, but their utility is compromised by their propensity to aggregate during manufacture and storage. As aggregation can be triggered by non-native states, whose population is not necessarily related to thermodynamic stability, prediction of poorly-behaving biologics is difficult, and searching for sequ...
Article
Full-text available
Aggregation of human α-synuclein (αSyn) is linked to Parkinson’s disease (PD) pathology. The central region of the αSyn sequence contains the non-amyloid β-component (NAC) crucial for aggregation. However, how NAC flanking regions modulate αSyn aggregation remains unclear. Using bioinformatics, mutation and NMR, we identify a 7-residue sequence, na...
Article
Full-text available
Protein aggregation occurs through a variety of mechanisms, initiated by the unfolded, non-native, or even the native state itself. Understanding the molecular mechanisms of protein aggregation is challenging, given the array of competing interactions that control solubility, stability, cooperativity and aggregation propensity. An array of methods...
Chapter
Single-molecule techniques provide insights into the heterogeneity and dynamics of ensembles and enable the extraction of mechanistic information that is complementary to high-resolution structural techniques. Here, we describe the application of single-molecule Förster resonance energy transfer to study the dynamics of integral membrane protein co...
Article
Full-text available
Amyloidogenesis is significant in both protein function and pathology. Amyloid formation of folded, globular proteins is commonly initiated by partial or complete 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 immunoglobuli...
Preprint
Full-text available
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, s...
Article
Full-text available
Differences in conformational dynamics between two full-length monoclonal antibodies have been probed in detail using Fast Photochemical Oxidation of Proteins (FPOP) followed by proteolysis and LC-ESI-MS/MS analyses. FPOP uses hydroxyl radical labelling to probe the surface-accessible regions of proteins and has the advantage that the resulting cov...
Preprint
The β-barrel assembly machinery (BAM) inserts outer membrane β-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 act...
Preprint
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 β2...
Preprint
Full-text available
The Sec translocon is a transmembrane assembly highly conserved among all forms of life as the principal route for transport of polypeptides across or into lipid bilayers. In bacteria translocation involves allosteric communication between the membrane pore SecYEG and the associated SecA ATPase. Using singlemolecule fluorescence we reveal that slow...
Article
Full-text available
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 character...
Article
Full-text available
In recent years our understanding of amyloid structure has been revolutionised by innovations in cryo-electron microscopy, electron diffraction and solid-state NMR. These techniques have yielded high-resolution structures of fibrils isolated from patients with neurodegenerative disease, as well as those formed from amyloidogenic proteins in vitro....
Article
Full-text available
Understanding the structural mechanism by which proteins and peptides aggregate is crucial, given the role of fibrillar aggregates in debilitating amyloid diseases and bioinspired materials. Yet, this is a major challenge as the assembly involves multiple heterogeneous and transient intermediates. Here, we analyze the co-aggregation of A 40 and A...
Article
Full-text available
Amyloid deposition of wild-type human β2-microglobulin (WT-hβ2m) in the joints of long-term hemodialysis patients is the hallmark of dialysis-related amyloidosis (DRA). In vitro, WT-hβ2m does not form amyloid fibrils at physiological pH and temperature unless co-solvents or other reagents are added. Therefore, understanding how fibril formation is...
Article
Full-text available
The nascent polypeptide-associated complex (NAC) is a conserved ribosome-associated protein biogenesis factor. Whether NAC exerts chaperone activity and whether this function is restricted to de novo protein synthesis is unknown. Here, we demonstrate that NAC directly exerts chaperone activity toward structurally diverse model substrates including...
Article
Full-text available
Chemical cross-linking mass-spectrometry (XL-MS) represents a powerful methodology to map ligand/biomacromolecule interactions, particularly where conventional methods such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy or cryo-electron microscopy (EM) are not feasible. In this manuscript, we describe the design and synthes...
Article
SurA is a conserved ATP-independent periplasmic chaperone involved in the biogenesis of outer-membrane proteins (OMPs). Escherichia coli SurA has a core domain and two peptidylprolyl isomerase (PPIase) domains, the role(s) of which remain unresolved. Here we show that while SurA homologues in early proteobacteria typically contain one or no PPIase...
Article
Full-text available
The aggregation of monomeric amyloid β protein (Aβ) peptide into oligomers and amyloid fibrils in the mammalian brain is associated with Alzheimer’s disease. Insight into the thermodynamic stability of the Aβ peptide in different polymeric states is fundamental to defining and predicting the aggregation process. Experimental determination of Aβ the...
Article
Full-text available
Analysing protein complexes by chemical crosslinking‐mass spectrometry (XL‐MS) is limited by the side‐chain reactivities and sizes of available crosslinkers, their slow reaction rates, and difficulties in crosslink enrichment, especially for rare, transient or dynamic complexes. Here we describe two new XL reagents that incorporate a methanethiosul...
Article
Full-text available
Analysing protein complexes by chemical crosslinking‐mass spectrometry (XL‐MS) is limited by the side‐chain reactivities and sizes of available crosslinkers, their slow reaction rates, and difficulties in crosslink enrichment, especially for rare, transient or dynamic complexes. Here we describe two new XL reagents that incorporate a methanethiosul...
Article
Full-text available
All amyloid fibrils contain a cross-β fold. How this structure differs in fibrils formed from proteins associated with different diseases remains unclear. Here, we combine cryo-EM and MAS-NMR to determine the structure of an amyloid fibril formed in vitro from β 2-micro-globulin (β 2 m), the culprit protein of dialysis-related amyloidosis. The fibr...
Article
With amyloid diseases poised to become a major health burden in countries with ageing populations, diagnostic molecules that aid the detection of amyloid in vitro and in vivo are of considerable clinical value. Understanding how such ligands recognize their amyloid targets would help to design diagnostics that target specific amyloid types associat...
Article
The aggregation of proteins into amyloid fibrils and their deposition into plaques and intracellular inclusions is the hallmark of amyloid disease. The accumulation and deposition of amyloid fibrils, collectively known as amyloidosis, is associated with many pathological conditions that can be associated with ageing, such as Alzheimer disease, Park...
Article
Full-text available
Hydrogen deuterium exchange (HDX) coupled to mass spectrometry (MS) is a well-established technique employed in the field of structural MS to probe the solvent accessibility, dynamics and hydrogen bonding of backbone amides in proteins. By contrast, fast photochemical oxidation of proteins (FPOP) uses hydroxyl radicals, liberated from the photolysi...
Article
Richard Nelson Perham, FRS, FMedSci, FRSA, was a British professor of structural biochemistry. He undertook his academic career at the University of Cambridge, holding positions as lecturer, reader, chair and head of the Department of Biochemistry, as well as becoming Master of St John's College. Perham published close to 300 scientific papers on p...
Article
Full-text available
Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. In bacteria, this is mostly mediated by the conserved SecYEG complex, driven through rounds of ATP hydrolysis by the cytoplasmic SecA, and the trans-membrane proton motive force. We have used single molecule techniques...