Portrait of Dr Wei-Feng Xue

Dr Wei-Feng Xue

Senior Lecturer in Chemical Biology
Director of Biochemistry Programme

About

What are the mechanisms that govern the formation of amyloid protein structures associated with human diseases such as Alzheimer’s disease, Parkinson’s disease, type 2 diabetes, Prion diseases and systemic amyloidosis? Why are some amyloid associated with devastating diseases while others are tolerated by cells or even perform functions important for life? These questions of fundamental biological importance are the focus of the research in the Xue laboratory.
Dr Wei-Feng Xue joined the school of Biosciences in 2011. He received his PhD degree in Physical Chemistry on research regarding protein-protein, protein-ligand and allosteric interactions in Prof. Sara Linse’s group at Lund University in Sweden in 2006. He then went on to become a postdoctoral fellow in the laboratory of Prof. Sheena Radford FRS at the Astbury Centre for Structural Molecular Biology at the University of Leeds on research topics concerning the mechanism and the biological impact of amyloid assembly. His research interests include supramolecular protein assembly, protein folding and misfolding, amyloid and prions, and AFM imaging.
Dr Wei-Feng Xue is a member of the Kent Fungal Group, and the Industrial Biotechnology Centre 

ORCID: orcid.org/0000-0002-6504-0404

Research interests

Amyloid structures consist of highly ordered forms of protein assembled from whole or parts of normal soluble proteins or peptides of diverse amino acid sequences. The devastating human diseases associated with amyloid, such as Alzheimer's disease, Creutzfeldt-Jakob (CJD prion disease), Huntington's disease, Parkinson disease, type II diabetes mellitus, and systemic amyloidosis, are linked to the way the amyloid structures are assembled and deposited in the brain or in other parts of the human body. But far from all amyloid assemblies are disease-associated, as some amyloid fibrils have also been recognised as a class of functional protein assemblies, which can play a number of important roles in bacteria, yeast and humans. A sub-class of amyloid can spread between organisms by forming small seeds through the breakage of larger fibrils. These are called prions, and they exist in humans where they cause prion diseases such as CJD. In yeast, prions confer special cellular properties in yeast cells that are passed on from generation to generation, as a form of epigenetic or 'protein gene'. Amyloid fibrils are defined by their cross-beta core structure, where continuous beta-sheets run through the core of amyloid fibrils perpendicularly to the fibril axis.
My research is focused on resolving the fundamental mechanisms that govern the formation and the molecular lifecycle of amyloid protein aggregates. The long-term research vision in my lab is to fully understand the assembly of protein fibrils, as well as how different mechanisms involved in amyloid assembly are linked to the disease-associated properties and useful biological functions of amyloid.

Teaching

Programme director for Biochemistry
First year 

  • BI321/BI3210: Biological Chemistry A (Module convenor) 
  • BI322/BI3220: Biological Chemistry B (Module convenor) 

Second year 

  • BI520: Metabolism and Metabolic Disease 
  • BI532: Skills for Bioscientists 2 

Third year 

  • BI600: Biology project 
  • BI629: Proteins: Structure and Function 

MSc 

  • BI852: Advanced Analytical and Emerging Technologies for Biotechnology and Bioengineers MSc by Research projects 

Supervision

We are currently looking for enthusiastic and motivated postgraduate students (MSc by research and PhD) as well as postdoctoral researchers intent on securing own fellowships to join our lab. If you are interested in the research in my lab, please contact: w.f.xue@kent.ac.uk Potential project titles and descriptions listed below, all projects will incur additional research costs of £1500.

Structural biology of amyloid aggregates 

A number of human disorders, for example Alzheimer’s disease (AD), Parkinson's disease (PD), type 2 diabetes, and transmissible spongiform emcephalopathies (TSEs), are associated with the abnormal folding and assembly of proteins. The net result of this misfolding is the formation of large insoluble protein deposits and small toxic and possibly transmissible protein particles in a state called amyloid. Amyloid diseases account for increasing medical and social importance, for example, more than half million people are suffering from AD in the UK alone, and PD affects about 1% of the population over the age of 60. The aim of this project is to identify and understand the structural organisation of amyloid aggregates using nano-scale imaging methods.
Additional research costs: £1500  

The molecular life-cycle of infectious prion particles
Why are some amyloid aggregates highly infectious prion particles while others are less infectious or even inert? This project aims to answer this fundamental question by investigating the molecular processes involved in the formation, growth and division of the prion protein Sup35. 
Additional research costs: £1500

Investigating the nano-scale properties of amyloid assembly 
Detailed characterisation of amyloid fibrils of different origins has revealed incredibly strong structures that are commonly only tens of nanometres thick but many micrometres long. The unusual physical characteristics of amyloid fibrils mean that they have also the potential to become strong and stable engineered nanomaterials. Here, using atomic force microscopy imaging approach (AFM), we are investigating the physical and mechanical properties of in vitro formed amyloid fibrils.
Additional research costs: £1500  

Synthetic biology approach to self-assembled fibrous bio-materials
The aims for this project is to design and produce functional amyloid fibrils displaying a selection of enzymes or small molecule/metal binding motifs, and to evaluate the structure and function of these fibrils.
Additional research costs: £1500   

Computational structural biology of amyloid aggregates
The aim of this project is to identify and understand the structural organisation of amyloid aggregates using nano-scale imaging methods and in this computational project, image and assembly kinetics data will be analysed in order to characterise and compare the molecular structures involved in the formation, growth and the division of amyloid aggregates grown from disease associated as well as biologically functional amyloidogenic proteins.
Additional research costs: £1200  

Professional

  • Editorial Board Member: Scientific Reports 
  • Editorial Board Member: Frontiers in Molecular Biosciences

Publications

Article

  • Miller, C. et al. (2018). A cell culture platform for Cryptosporidium that enables long-term cultivation and new tools for the systematic investigation of its biology. International Journal for Parasitology [Online] 48:197-201. Available at: https://doi.org/10.1016/j.ijpara.2017.10.001.
    Cryptosporidium parasites are a major cause of diarrhoea that pose a particular threat to children in developing areas and immunocompromised individuals. Curative therapies and vaccines are lacking, mainly due to lack of a long-term culturing system of this parasite. Here, we show that COLO-680N cells infected with two different Cryptosporidium parvum strains produce sufficient infectious oocysts to infect subsequent cultures, showing a substantial fold increase in production, depending on the experiment, over the most optimistic HCT-8 models. Oocyst identity was confirmed using a variety of microscopic- and molecular-based methods. This culturing system will accelerate research on Cryptosporidium and the development of anti-Cryptosporidium drugs.
  • Galloway, J. et al. (2018). Bioinspired Silicification Reveals Structural Detail in Self-Assembled Peptide Cages. ACS Nano [Online] 12:1420-1432. Available at: http://dx.doi.org/10.1021/acsnano.7b07785.
    Understanding how molecules in self-assembled soft-matter nanostructures are organized is essential for improving the design of next-generation nanomaterials. Imaging these assemblies can be challenging and usually requires processing, e.g., staining or embedding, which can damage or obscure features. An alternative is to use bioinspired mineralization, mimicking how certain organisms use biomolecules to template mineral formation. Previously, we have reported the design and characterization of Self-Assembled peptide caGEs (SAGEs) formed from de novo peptide building blocks. In SAGEs, two complementary, 3-fold symmetric, peptide hubs combine to form a hexagonal lattice, which curves and closes to form SAGE nanoparticles. As hexagons alone cannot tile onto spheres, the network must also incorporate nonhexagonal shapes. While the hexagonal ultrastructure of the SAGEs has been imaged, these defects have not been observed. Here, we show that positively charged SAGEs biotemplate a thin, protective silica coating. Electron microscopy shows that these SiO2-SAGEs do not collapse, but maintain their 3D shape when dried. Atomic force microscopy reveals a network of hexagonal and irregular features on the SiO2-SAGE surface. The dimensions of these (7.2 nm ± 1.4 nm across, internal angles 119.8° ± 26.1°) are in accord with the designed SAGE network and with coarse-grained modeling of the SAGE assembly. The SiO2-SAGEs are permeable to small molecules (<2 nm), but not to larger biomolecules (>6 nm). Thus, bioinspired silicification offers a mild technique that preserves soft-matter nanoparticles for imaging, revealing structural details <10 nm in size, while also maintaining desirable properties, such as permeability to small molecules.
  • Lee, M. et al. (2017). Engineered synthetic scaffolds for organizing proteins within the bacterial cytoplasm. Nature Chemical Biology [Online] 14:142-147. Available at: https://doi.org/10.1038/nchembio.2535.
    We have developed a system for producing a supramolecular scaffold that permeates the entire Escherichia coli cytoplasm. This cytoscaffold is constructed from a three-component system comprising a bacterial microcompartment shell protein and two complementary de novo coiled-coil peptides. We show that other proteins can be targeted to this intracellular filamentous arrangement. Specifically, the enzymes pyruvate decarboxylase and alcohol dehydrogenase have been directed to the filaments, leading to enhanced ethanol production in these engineered bacterial cells compared to those that do not produce the scaffold. This is consistent with improved metabolic efficiency through enzyme colocation. Finally, the shell-protein scaffold can be directed to the inner membrane of the cell, demonstrating how synthetic cellular organization can be coupled with spatial optimization through in-cell protein design. The cytoscaffold has potential in the development of next-generation cell factories, wherein it could be used to organize enzyme pathways and metabolite transporters to enhance metabolic flux.
  • Marchante, R. et al. (2017). The physical dimensions of amyloid aggregates control their infective potential as prion particles. eLife [Online] 6:e27109. Available at: https://doi.org/10.7554/eLife.27109.001.
    Transmissible amyloid particles called prions are associated with infectious prion diseases in mammals and inherited phenotypes in yeast. All amyloid aggregates can give rise to potentially infectious seeds that accelerate their growth. Why some amyloid seeds are highly infectious prion particles while others are less infectious or even inert, is currently not understood. To address this question, we analyzed the suprastructure and dimensions of synthetic amyloid fibrils assembled from the yeast (Saccharomyces cerevisiae) prion protein Sup35NM. We then quantified the ability of these particles to induce the [PSI(+)] prion phenotype in cells. Our results show a striking relationship between the length distribution of the amyloid fibrils and their ability to induce the heritable [PSI(+)] prion phenotype. Using a simple particle size threshold model to describe transfection activity, we explain how dimensions of amyloid fibrils are able to modulate their infectious potential as prions.
  • Eugène, S. et al. (2016). Insights into the variability of nucleated amyloid polymerization by a minimalistic model of stochastic protein assembly. The Journal of Chemical Physics [Online] 144:175101. Available at: http://doi.org/10.1063/1.4947472.
    Self-assembly of proteins into amyloid aggregates is an important biological phenomenon associated with human diseases such as Alzheimer’s disease. Amyloid brils also have potential applications in nano-engineering of biomaterials. The kinetics of amyloid assembly show an exponential growth phase preceded by a lag phase, variable in duration as seen in bulk experiments and experiments that mimic the small volumes of cells. Here, to investigate the origins and the properties of the observed variability in the lag phase of amyloid assembly currently not accounted for by deterministic nucleation dependent mechanisms, we formulate a new stochastic minimal model that is capable of describing the characteristics of amyloid growth curves despite its simplicity. We then solve the stochastic di erential equations of our model and give mathematical proof of a central limit theorem for the sample growth trajectories of the nucleated aggregation process. These results give an asymptotic description for our simple model, from which closed form analytical results capable of describing and predicting the variability of nucleated amyloid assembly were derived. We also demonstrate the application of our results to inform experiments in a conceptually friendly and clear fashion. Our model o ers a new perspective and paves the way for a new and e cient approach on extracting vital information regarding the key initial events of amyloid formation.
  • Mayer, M. et al. (2016). Effect of bio-engineering on size, shape, composition and rigidity of bacterial microcompartments. Scientific Reports [Online] 6:36899. Available at: http://dx.doi.org/10.1038/srep36899.
  • Al-Hilaly, Y. et al. (2016). The involvement of dityrosine crosslinking in ?-synuclein assembly and deposition in Lewy Bodies in Parkinson's disease. Scientific Reports [Online] 6:39171. Available at: http://dx.doi.org/10.1038/srep39171.
    Parkinson's disease (PD) is characterized by intracellular, insoluble Lewy bodies composed of highly stable ?-synuclein (?-syn) amyloid fibrils. ?-synuclein is an intrinsically disordered protein that has the capacity to assemble to form ?-sheet rich fibrils. Oxidiative stress and metal rich environments have been implicated in triggering assembly. Here, we have explored the composition of Lewy bodies in post-mortem tissue using electron microscopy and immunogold labeling and revealed dityrosine crosslinks in Lewy bodies in brain tissue from PD patients. In vitro, we show that dityrosine cross-links in ?-syn are formed by covalent ortho-ortho coupling of two tyrosine residues under conditions of oxidative stress by fluorescence and confirmed using mass-spectrometry. A covalently cross-linked dimer isolated by SDS-PAGE and mass analysis showed that dityrosine dimer was formed via the coupling of Y39-Y39 to give a homo dimer peptide that may play a key role in formation of oligomeric and seeds for fibril formation. Atomic force microscopy analysis reveals that the covalent dityrosine contributes to the stabilization of ?-syn assemblies. Thus, the presence of oxidative stress induced dityrosine could play an important role in assembly and toxicity of ?-syn in PD.
  • Smith, R. et al. (2015). Analysis of Toxic Amyloid Fibril Interactions at Natively Derived Membranes by Ellipsometry. PLOS ONE [Online] 10:e0132309. Available at: http://doi.org/10.1371/journal.pone.0132309.
    There is an ongoing debate regarding the culprits of cytotoxicity associated with amyloid disorders. Although small pre-fibrillar amyloid oligomers have been implicated as the primary toxic species, the fibrillar amyloid material itself can also induce cytotoxicity. To investigate membrane disruption and cytotoxic effects associated with intact and fragmented fibrils, the novel in situ spectroscopic technique of Total Internal Reflection Ellipsometry (TIRE) was used. Fibril lipid interactions were monitored using natively derived whole cell membranes as a model of the in vivo environment. We show that fragmented fibrils have an increased ability to disrupt these natively derived membranes by causing a loss of material from the deposited surface when compared with unfragmented fibrils. This effect was corroborated by observations of membrane disruption in live cells, and by dye release assay using synthetic liposomes. Through these studies we demonstrate the use of TIRE for the analysis of protein-lipid interactions on natively derived lipid surfaces, and provide an explanation on how amyloid fibrils can cause a toxic gain of function, while entangled amyloid plaques exert minimal biological activity.
  • Xue, W. (2015). Nucleation: The Birth of a New Protein Phase. Biophysical Journal [Online] 109:1999-2000. Available at: http://dx.doi.org/10.1016/j.bpj.2015.10.011.
  • Lawrence, A. et al. (2014). Solution Structure of a Bacterial Microcompartment Targeting Peptide and Its Application in the Construction of an Ethanol Bioreactor. ACS Synthetic Biology [Online] 3:454-465. Available at: http://dx.doi.org/10.1021/sb4001118.
    Targeting of proteins to bacterial microcompartments (BMCs) is mediated by an 18-amino-acid peptide sequence. Herein, we report the solution structure of the N-terminal targeting peptide (P18) of PduP, the aldehyde dehydrogenase associated with the 1,2-propanediol utilization metabolosome from Citrobacter freundii. The solution structure reveals the peptide to have a well-defined helical conformation along its whole length. Saturation transfer difference and transferred NOE NMR has highlighted the observed interaction surface on the peptide with its main interacting shell protein, PduK. By tagging both a pyruvate decarboxylase and an alcohol dehydrogenase with targeting peptides, it has been possible to direct these enzymes to empty BMCs in vivo and to generate an ethanol bioreactor. Not only are the purified, redesigned BMCs able to transform pyruvate into ethanol efficiently, but the strains containing the modified BMCs produce elevated levels of alcohol.
  • Jakhria, T. et al. (2014). Beta2-Microglobulin Amyloid Fibrils Are Nanoparticles That Disrupt Lysosomal Membrane Protein Trafficking and Inhibit Protein Degradation by Lysosomes. Journal of Biological Chemistry [Online] 289:35781-35794. Available at: http://dx.doi.org/10.1074/jbc.M114.586222.
  • Goodchild, S. et al. (2014). ?2-Microglobulin Amyloid Fibril-Induced Membrane Disruption Is Enhanced by Endosomal Lipids and Acidic pH. PLoS One [Online] 9:e104492. Available at: http://dx.doi.org/10.1371/journal.pone.0104492.
    Although the molecular mechanisms underlying the pathology of amyloidoses are not well understood, the interaction between amyloid proteins and cell membranes is thought to play a role in several amyloid diseases. Amyloid fibrils of ?2-microglobulin (?2m), associated with dialysis-related amyloidosis (DRA), have been shown to cause disruption of anionic lipid bilayers in vitro. However, the effect of lipid composition and the chemical environment in which ?2m-lipid interactions occur have not been investigated previously. Here we examine membrane damage resulting from the interaction of ?2m monomers and fibrils with lipid bilayers. Using dye release, tryptophan fluorescence quenching and fluorescence confocal microscopy assays we investigate the effect of anionic lipid composition and pH on the susceptibility of liposomes to fibril-induced membrane damage. We show that ?2m fibril-induced membrane disruption is modulated by anionic lipid composition and is enhanced by acidic pH. Most strikingly, the greatest degree of membrane disruption is observed for liposomes containing bis(monoacylglycero)phosphate (BMP) at acidic pH, conditions likely to reflect those encountered in the endocytic pathway. The results suggest that the interaction between ?2m fibrils and membranes of endosomal origin may play a role in the molecular mechanism of ?2m amyloid-associated osteoarticular tissue destruction in DRA.
  • Marshall, K. et al. (2014). The relationship between amyloid structure and cytotoxicity. Prion [Online] 8:192-196. Available at: http://dx.doi.org/10.4161/pri.28860.
    Self-assembly of proteins and peptides into amyloid structures has been the subject of intense and focused research due to their association with neurodegenerative, age-related human diseases and transmissible prion diseases in humans and mammals. Of the disease associated amyloid assemblies, a diverse array of species, ranging from small oligomeric assembly intermediates to fibrillar structures, have been shown to have toxic potential. Equally, a range of species formed by the same disease associated amyloid sequences have been found to be relatively benign under comparable monomer equivalent concentrations and conditions. In recent years, an increasing number of functional amyloids have also been found. These developments show that not all amyloid structures are generically toxic to cells. Given these observations, it is important to understand why amyloid structures may encode such varied toxic potential despite sharing a common core molecular architecture. Here, we discuss possible links between different aspects of amyloidogenic structures and assembly mechanisms with their varied functional effects. We propose testable hypotheses for the relationship between amyloid structure and its toxic potential in the context of recent reports on amyloid sequence, structure, and toxicity relationships.
  • Tuite, M., Howard, M. and Xue, W. (2014). Dynamic Prions Revealed by Magic. Chemistry & Biology [Online] 21:172-173. Available at: http://dx.doi.org/10.1016/j.chembiol.2014.02.001.
    Prion proteins can be propagated as amyloid fibrils with several different conformational variants. By providing structural information at atomic level for two such variants of a yeast prion, Frederick and colleagues, in this issue of Chemistry & Biology, reveal how conformational flexibility can generate pheno- typic diversity.
  • Sheynis, T. et al. (2013). Aggregation modulators interfere with membrane interactions of beta2-microglobulin fibrils. Biophysical Journal [Online] 105:745-755. Available at: http://dx.doi.org/10.1016/j.bpj.2013.06.015.
    Amyloid fibril accumulation is a pathological hallmark of several devastating disorders, including Alzheimer's disease, prion diseases, type II diabetes, and others. Although the molecular factors responsible for amyloid pathologies have not been deciphered, interactions of misfolded proteins with cell membranes appear to play important roles in these disorders. Despite increasing evidence for the involvement of membranes in amyloid-mediated cytotoxicity, the pursuit for therapeutic strategies has focused on preventing self-assembly of the proteins comprising the amyloid plaques. Here we present an investigation of the impact of fibrillation modulators upon membrane interactions of ?2-microglobulin (?2m) fibrils. The experiments reveal that polyphenols (epigallocatechin gallate, bromophenol blue, and resveratrol) and glycosaminoglycans (heparin and heparin disaccharide) differentially affect membrane interactions of ?2m fibrils measured by dye-release experiments, fluorescence anisotropy of labeled lipid, and confocal and cryo-electron microscopies. Interestingly, whereas epigallocatechin gallate and heparin prevent membrane damage as judged by these assays, the other compounds tested had little, or no, effect. The results suggest a new dimension to the biological impact of fibrillation modulators that involves interference with membrane interactions of amyloid species, adding to contemporary strategies for combating amyloid diseases that focus on disruption or remodeling of amyloid aggregates.
  • Xue, W. and Radford, S. (2013). An Imaging and Systems Modeling Approach to Fibril Breakage Enables Prediction of Amyloid Behavior. Biophysical Journal [Online] 105:2811-2819. Available at: http://dx.doi.org/10.1016/j.bpj.2013.10.034.
    Delineating the nanoscale properties and the dynamic assembly and disassembly behaviors of amyloid fibrils is key for technological applications that use the material properties of amyloid fibrils, as well as for developing treatments of amyloid-associated disease. However, quantitative mechanistic understanding of the complex processes involving these heterogeneous supramolecular systems presents challenges that have yet to be resolved. Here, we develop an approach that is capable of resolving the time dependence of fibril particle concentration, length distribution, and length and position dependence of fibril fragmentation rates using a generic mathematical framework combined with experimental data derived from atomic force microscopy analysis of fibril length distributions. By application to amyloid assembly of ?2-microglobulin in vitro under constant mechanical stirring, we present a full description of the fibril fragmentation and growth behavior, and demonstrate the predictive power of the approach in terms of the samples’ fibril dimensions, fibril load, and their efficiency to seed the growth of new amyloid fibrils. The approach developed offers opportunities to determine, quantify, and predict the course and the consequences of amyloid assembly.
  • Milanesi, L. et al. (2012). Direct three-dimensional visualization of membrane disruption by amyloid fibrils. Proceedings of the National Academy of Sciences of the United States of America 109:20455-60.
    Protein misfolding and aggregation cause serious degenerative conditions such as Alzheimer's, Parkinson, and prion diseases. Damage to membranes is thought to be one of the mechanisms underlying cellular toxicity of a range of amyloid assemblies. Previous studies have indicated that amyloid fibrils can cause membrane leakage and elicit cellular damage, and these effects are enhanced by fragmentation of the fibrils. Here we report direct 3D visualization of membrane damage by specific interactions of a lipid bilayer with amyloid-like fibrils formed in vitro from ?(2)-microglobulin (?(2)m). Using cryoelectron tomography, we demonstrate that fragmented ?(2)m amyloid fibrils interact strongly with liposomes and cause distortions to the membranes. The normally spherical liposomes form pointed teardrop-like shapes with the fibril ends seen in proximity to the pointed regions on the membranes. Moreover, the tomograms indicated that the fibrils extract lipid from the membranes at these points of distortion by removal or blebbing of the outer membrane leaflet. Tiny (15-25 nm) vesicles, presumably formed from the extracted lipids, were observed to be decorating the fibrils. The findings highlight a potential role of fibrils, and particularly fibril ends, in amyloid pathology, and report a previously undescribed class of lipid-protein interactions in membrane remodelling.
  • Strawn, R. et al. (2011). Synergy of molecular dynamics and isothermal titration calorimetry in studies of allostery. Methods in Enzymology [Online] 492:151-88. Available at: http://dx.doi.org/10.1016/B978-0-12-381268-1.00017-3.
    Despite decades of intensive study, allosteric effects have eluded an intellectually satisfying integrated understanding that includes a description of the reaction coordinate in terms of species distributions of structures and free energy levels in the conformational ensemble. This chapter illustrates a way to fill this gap by interpreting thermodynamic and structural results through the lens of molecular dynamics simulation analysis to link atomic-level detail with global response. In this synergistic approach molecular dynamics forms an integral part of a feedback loop of hypothesis, experimental design, and interpretation that conforms to the scientific method.
  • Xue, W. et al. (2010). Fibril fragmentation in amyloid assembly and cytotoxicity: When size matters. Prion [Online] 4:20-25. Available at: http://dx.doi.org/10.4161/pri.4.1.11378.
    Amyloid assemblies are associated with several debilitating human disorders. Understanding the intra- and extracellular assembly of normally soluble proteins and peptides into amyloid aggregates and how they disrupt normal cellular functions is therefore of paramount importance. In a recent report, we demonstrated a striking relationship between reduced fibril length caused by fibril fragmentation and enhanced ability of fibril samples to disrupt membranes and to reduce cell viability. These findings have important implications for our understanding of amyloid disease in that changes in the physical dimensions of fibrils, without parallel changes in their composition or molecular architecture, could be sufficient to alter the biological responses to their presence. These conclusions provide a new hypothesis that the physical dimensions and surface interactions of fibrils play key roles in amyloid disease. Controlling fibril length and stability toward fracturing, and thereby the biological availability of fibril material, may provide a new target for future therapeutic strategies towards combating amyloid disease
  • Platt, G. et al. (2009). Probing Dynamics within Amyloid Fibrils Using a Novel Capping Method. Angewandte Chemie International Edition [Online] 48:5705-5707. Available at: http://dx.doi.org/10.1002/anie.200901343.
  • Xue, W. et al. (2009). Fibril Fragmentation Enhances Amyloid Cytotoxicity. Journal of Biological Chemistry [Online] 284:34272-34282. Available at: http://dx.doi.org/10.1074/jbc.M109.049809.
    Fibrils associated with amyloid disease are molecular assemblies of key biological importance, yet how cells respond to the presence of amyloid remains unclear. Cellular responses may not only depend on the chemical composition or molecular properties of the amyloid fibrils, but their physical attributes such as length, width, or surface area may also play important roles. Here, we report a systematic investigation of the effect of fragmentation on the structural and biological properties of amyloid fibrils. In addition to the expected relationship between fragmentation and the ability to seed, we show a striking finding that fibril length correlates with the ability to disrupt membranes and to reduce cell viability. Thus, despite otherwise unchanged molecular architecture, shorter fibrillar samples show enhanced cytotoxic potential than their longer counterparts. The results highlight the importance of fibril length in amyloid disease, with fragmentation not only providing a mechanism by which fibril load can be rapidly increased but also creating fibrillar species of different dimensions that can endow new or enhanced biological properties such as amyloid cytotoxicity.
  • Xue, W. et al. (2009). Role of protein surface charge in monellin sweetness. Biochimica Et Biophysica Acta-Proteins and Proteomics [Online] 1794:410-420. Available at: http://dx.doi.org/10.1016/j.bbapap.2008.11.008.
    A small number of proteins have the unusual property of tasting intensely sweet. Despite many studies aimed at identifying their sweet taste determinants, the molecular basis of protein sweetness is not fully understood. Recent mutational studies of monellin have implicated positively charged residues in sweetness. In the present work, the effect of overall net charge was investigated using the complementary approach of negative charge alterations. Multiple substitutions of Asp/Asn and Glu/Gln residues radically altered the surface charge of single-chain monellin by removing six negative charges or adding four negative charges. Biophysical characterization using circular dichroism, fluorescence, and two-dimensional NMR demonstrates that the native fold of monellin is preserved in the variant proteins under physiological solution conditions although their stability toward chemical denaturation is altered. A human taste test was employed to determine the sweetness detection threshold of the variants. Removal of negative charges preserves monellin sweetness, whereas added negative charge has a large negative impact on sweetness. Meta-analysis of published charge variants of monellin and other sweet proteins reveals a general trend toward increasing sweetness with increasing positive net charge. Structural mapping of monellin variants identifies a hydrophobic surface predicted to face the receptor where introduced positive or negative charge reduces sweetness, and a polar surface where charges modulate long-range electrostatic complementarity.
  • Bauer, M., Xue, W. and Linse, S. (2009). Protein GB1 folding and assembly from structural elements. International Journal of Molecular Sciences [Online] 10:1552-1566. Available at: http://dx.doi.org/10.3390/ijms10041552.
    Folding of the Protein G B1 domain (PGB1) shifts with increasing salt concentration from a cooperative assembly of inherently unstructured subdomains to an assembly of partly pre-folded structures. The salt-dependence of pre-folding contributes to the stability minimum observed at physiological salt conditions. Our conclusions are based on a study in which the reconstitution of PGB1 from two fragments was studied as a function of salt concentrations and temperature using circular dichroism spectroscopy. Salt was found to induce an increase in beta-hairpin structure for the C-terminal fragment (residues 41 - 56), whereas no major salt effect on structure was observed for the isolated N-terminal fragment (residues 1 - 41). In line with the increasing evidence on the interrelation between fragment complementation and stability of the corresponding intact protein, we also find that salt effects on reconstitution can be predicted from salt dependence of the stability of the intact protein. Our data show that our variant (which has the mutations T2Q, N8D, N37D and reconstitutes in a manner similar to the wild type) displays the lowest equilibrium association constant around physiological salt concentration, with higher affinity observed both at lower and higher salt concentration. This corroborates the salt effects on the stability towards denaturation of the intact protein, for which the stability at physiological salt is lower compared to both lower and higher salt concentrations. Hence we conclude that reconstitution reports on molecular factors that govern the native states of proteins.
  • Xue, W., Homans, S. and Radford, S. (2009). Amyloid fibril length distribution quantified by atomic force microscopy single-particle image analysis. Protein Engineering Design and Selection [Online] 22:489-496. Available at: http://dx.doi.org/10.1093/protein/gzp026.
    Amyloid fibrils are proteinaceous nano-scale linear aggregates. They are of key interest not only because of their association with numerous disorders, such as type II diabetes mellitus, Alzheimer's and Parkinson's diseases, but also because of their potential to become engineered high-performance nano-materials. Methods to characterise the length distribution of nano-scale linear aggregates such as amyloid fibrils are of paramount importance both in understanding the biological impact of these aggregates and in controlling their mechanical properties as potential nano-materials. Here, we present a new quantitative approach to the determination of the length distribution of amyloid fibrils using tapping-mode atomic force microscopy. The method described employs single-particle image analysis corrected for the length-dependent bias that is a common problem associated with surface-based imaging techniques. Applying this method, we provide a detailed characterisation of the length distribution of samples containing long-straight fibrils formed in vitro from ?2-microglobulin. The results suggest that the Weibull distribution is a suitable model in describing fibril length distributions, and reveal that fibril fragmentation is an important process even under unagitated conditions. These results demonstrate the significance of quantitative length distribution measurements in providing important new information regarding amyloid assembly
  • Xue, W., Homans, S. and Radford, S. (2008). Systematic analysis of nucleation-dependent polymerization reveals new insights into the mechanism of amyloid self-assembly. Proceedings of the National Academy of Sciences of the United States of America [Online] 105:8926-8931. Available at: http://dx.doi.org/10.1073/pnas.0711664105.
    Self-assembly of misfolded proteins into ordered fibrillar aggregates known as amyloid results in numerous human diseases. Despite an increasing number of proteins and peptide fragments being recognised as amyloidogenic, how these amyloid aggregates assemble remains unclear. In particular, the identity of the nucleating species, an ephemeral entity that defines the rate of fibril formation, remains a key outstanding question. Here, we propose a new strategy for analyzing the self-assembly of amyloid fibrils involving global analysis of a large number of reaction progress curves and the subsequent systematic testing and ranking of a large number of possible assembly mechanisms. Using this approach, we have characterized the mechanism of the nucleation-dependent formation of ?2-microglobulin (?2m) amyloid fibrils. We show, by defining nucleation in the context of both structural and thermodynamic aspects, that a model involving a structural nucleus size approximately the size of a hexamer is consistent with the relatively small concentration dependence of the rate of fibril formation, contrary to expectations based on simpler theories of nucleated assembly. We also demonstrate that fibril fragmentation is the dominant secondary process that produces higher apparent cooperatively in fibril formation than predicted by nucleated assembly theories alone. The model developed is able to explain and predict the behavior of ?2m fibril formation and provides a rationale for explaining generic properties observed in other amyloid systems, such as fibril growth acceleration and pathway shifts under agitation
  • Linse, S. et al. (2007). Nucleation of protein fibrillation by nanoparticles. Proceedings of the National Academy of Sciences of the United States of America [Online] 104:8691-8696. Available at: http://dx.doi.org/10.1073/pnas.0701250104.
    Nanoparticles present enormous surface areas and are found to enhance the rate of protein fibrillation by decreasing the lag time for nucleation. Protein fibrillation is involved in many human diseases, including Alzheimer's, Creutzfeld-Jacob disease, and dialysis-related amyloidosis. Fibril formation occurs by nucleation-dependent kinetics, wherein formation of a critical nucleus is the key rate-determining step, after which fibrillation proceeds rapidly. We show that nanoparticles (copolymer particles, cerium oxide particles, quantum dots, and carbon nanotubes) enhance the probability of appearance of a critical nucleus for nucleation of protein fibrils from human beta(2)-microglobulin. The observed shorter lag (nucleation) phase depends on the amount and nature of particle surface. There is an exchange of protein between solution and nanoparticle surface, and beta(2)-microglobulin forms multiple layers on the particle surface, providing a locally increased protein concentration promoting oligomer formation. This and the shortened lag phase suggest a mechanism involving surface-assisted nucleation that may increase the risk for toxic cluster and amyloid formation. It also opens the door to new routes for the controlled self-assembly of proteins and peptides into novel nanomaterials.
  • Lindman, S. et al. (2006). Salting the charged surface: pH and salt dependence of protein G B1 stability. Biophysical Journal [Online] 90:2911-2921. Available at: http://dx.doi.org/10.1529/biophysj.105.071050.
    This study shows significant effects of protein surface charges on stability and these effects are not eliminated by salt screening. The stability for a variant of protein G B1 domain was studied in the pH-range of 1.5-11 at low, 0.15 M, and 2 M salt. The variant has three mutations, T2Q, N8D, and N37D, to guarantee an intact covalent chain at all pH values. The stability of the protein shows distinct pH dependence with the highest stability close to the isoelectric point. The stability is pH-dependent at all three NaCl concentrations, indicating that interactions involving charged residues are important at all three conditions. We find that 2 M salt stabilizes the protein at low pH (protein net charge is +6 and total number of charges is 6) but not at high pH (net charge is <or=-6 and total number of charges is >or=18). Furthermore, 0.15 M salt slightly decreases the stability of the protein over the pH range. The results show that a net charge of the protein is destabilizing and indicate that proteins contain charges for reasons other than improved stability. Salt seems to reduce the electrostatic contributions to stability under conditions with few total charges, but cannot eliminate electrostatic effects in highly charged systems.
  • Xue, W. et al. (2006). Intra- versus intermolecular interactions in monellin: contribution of surface charges to protein assembly. Journal of Molecular Biology [Online] 358:1244-1255. Available at: http://dx.doi.org/doi:10.1016/j.jmb.2006.02.069.
    The relative significance of weak non-covalent interactions in biological context has been much debated. Here, we have addressed the contribution of Coulombic interactions to protein stability and assembly experimentally. The sweet protein monellin, a non-covalently linked heterodimeric protein, was chosen for this study because of its ability to spontaneously reconstitute from separated fragments. The reconstitution of monellin mutants containing large surface charge perturbations was compared to the thermostability of structurally equivalent single-chain monellin containing the same sets of mutations under varying salt concentrations. The affinity between monellin fragments is found to correlate with the thermostability of single chain monellin, indicating the involvement of the same underlying Coulombic interactions. This confirms that there are no principal differences in the interactions involved in folding and binding. Based on comparison with a previous mutational study involving hydrophobic core residues, the relative contribution of Coulombic interactions to stability and affinity is modest. However, the Coulombic perturbations only affect the association rates of reconstitution in contrast to perturbations involving hydrophobic residues, which affect primarily the dissociation rates. These results indicate that Coulombic interactions are likely to be of main importance for the association of protein assembly, relevant for functions of proteins.
  • Jin, L. et al. (2005). Asymmetric allosteric activation of the symmetric ArgR hexamer. Journal of Molecular Biology [Online] 346:43-56. Available at: http://dx.doi.org/doi:10.1016/j.jmb.2004.11.031.
    Hexameric arginine repressor, ArgR, bound to L-arginine serves both as the master transcriptional repressor/activator at diverse regulons in a wide range of bacteria and as a required cofactor for resolution of ColE1 plasmid multimers. Multifunctional ArgR is thus unusual in possessing features of specific gene regulators, global regulators, and non-specific gene organizers; its closest functional analog is probably CAP, the cyclic AMP receptor/activator protein. Isothermal titration calorimetry, surface plasmon resonance, and proteolysis indicate that binding of a single L-argine [corrected] per ArgR hexamer triggers a global conformation [corrected] change and resets the affinities of the remaining five sites, making them 100-fold weaker. The analysis suggests a novel thermodynamic signature for this mechanism of activation.
  • Dell'Orco, D. et al. (2005). Electrostatic contributions to the kinetics and thermodynamics of protein assembly. Biophysical Journal [Online] 88:1991-2002. Available at: http://dx.doi.org/10.1529/biophysj.104.049189.
    The role of electrostatic interactions in the assembly of a native protein structure was studied using fragment complementation. Contributions of salt, pH, or surface charges to the kinetics and equilibrium of calbindin D(9k) reconstitution was measured in the presence of Ca(2+) using surface plasmon resonance and isothermal titration calorimetry. Whereas surface charge substitutions primarily affect the dissociation rate constant, the association rates are correlated with subdomain net charge in a way expected for Coulomb interactions. The affinity is reduced in all mutants, with the largest effect (260-fold) observed for the double mutant K25E+K29E. At low net charge, detailed charge distribution is important, and charges remote from the partner EF-hand have less influence than close ones. The effects of salt and pH on the reconstitution are smaller than mutational effects. The interaction between the wild-type EF-hands occurs with high affinity (K(A) = 1.3 x 10(10) M(-1); K(D) = 80 pM). The enthalpy of association is overall favorable and there appears to be a very large favorable entropic contribution from the desolvation of hydrophobic surfaces that become buried in the complex. Electrostatic interactions contribute significantly to the affinity between the subdomains, but other factors, such as hydrophobic interactions, dominate.
  • Xue, W., Carey, J. and Linse, S. (2004). Multi-method global analysis of thermodynamics and kinetics in reconstitution of monellin. Proteins [Online] 57:586-95. Available at: http://dx.doi.org/10.1002/prot.20241.
    Accurate and precise determinations of thermodynamic parameters of binding are important steps toward understanding many biological mechanisms. Here, a multi-method approach to binding analysis is applied and a detailed error analysis is introduced. Using this approach, the binding thermodynamics and kinetics of the reconstitution of the protein monellin have been quantitatively determined in detail by simultaneous analysis of data collected with fluorescence spectroscopy, surface plasmon resonance and isothermal titration calorimetry at 25 degrees C, pH 7.0 and 150 mM NaCl. Monellin is an intensely sweet protein composed of two peptide chains that form a single globular domain. The kinetics of the reconstitution reaction are slow, with an association rate constant, k(on) of 8.8 x 10(3) M(-1) s(-1) and a dissociation rate constant, k(off) of 3.1 x 10(-4) s(-1). The equilibrium constant K(A) is 2.8 x 10(7) M(-1) corresponding to a standard free energy of association, DeltaG degrees , of -42.5 kJ/mol. The enthalpic component, DeltaH degrees , is -18.7 kJ/mol and the entropic contribution, DeltaS degrees , is 79.8 J mol(-1) K(-1) (-TDeltaS degrees = -23.8 kJ/mol). The association of monellin is therefore a bimolecular intra-protein association whose energetics are slightly dominated by entropic factors.

Book section

  • Xue, W. (2013). Amyloid Fibril Length Quantification by Atomic Force Microscopy. in: Uversky, V. N. and Lyubchenko, Y. L. eds. Bio-Nanoimaging - Protein Misfolding & Aggregation. Elsevier, pp. 17-25. Available at: http://store.elsevier.com/Bio-nanoimaging/isbn-9780123944313/.

Image

  • Xue, W. and Miszkiewicz, J. (2013). Human femoral bone, AFM. [Online]. Available at: http://wellcomeimages.org/indexplus/image/B0009362.html.
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