Journal of the American Chemical Society, May 18, 2016
The balance between protein folding and misfolding is a crucial determinant of amyloid assembly. ... more The balance between protein folding and misfolding is a crucial determinant of amyloid assembly. Transient intermediates that are sparsely populated during protein folding have been identified as key players in amyloid aggregation. However, due to their ephemeral nature, structural characterization of these species remains challenging. Here, using the power of nonuniformly sampled NMR methods we investigate the folding pathway of amyloidogenic and nonamyloidogenic variants of β2-microglobulin (β2m) in atomic detail. Despite folding via common intermediate states, we show that the decreased population of the aggregation-prone ITrans state and population of a less stable, more dynamic species ablate amyloid formation by increasing the energy barrier for amyloid assembly. The results show that subtle changes in conformational dynamics can have a dramatic effect in determining whether a protein is amyloidogenic, without perturbation of the mechanism of protein folding.
Proceedings of the National Academy of Sciences of the United States of America, Jan 20, 2015
Amyloid disorders cause debilitating illnesses through the formation of toxic protein aggregates.... more Amyloid disorders cause debilitating illnesses through the formation of toxic protein aggregates. The mechanisms of amyloid toxicity and the nature of species responsible for mediating cellular dysfunction remain unclear. Here, using β2-microglobulin (β2m) as a model system, we show that the disruption of membranes by amyloid fibrils is caused by the molecular shedding of membrane-active oligomers in a process that is dependent on pH. Using thioflavin T (ThT) fluorescence, NMR, EM and fluorescence correlation spectroscopy (FCS), we show that fibril disassembly at pH 6.4 results in the formation of nonnative spherical oligomers that disrupt synthetic membranes. By contrast, fibril dissociation at pH 7.4 results in the formation of nontoxic, native monomers. Chemical cross-linking or interaction with hsp70 increases the kinetic stability of fibrils and decreases their capacity to cause membrane disruption and cellular dysfunction. The results demonstrate how pH can modulate the delete...
In the early stages of amyloid formation, heterogeneous populations of oligomeric species are gen... more In the early stages of amyloid formation, heterogeneous populations of oligomeric species are generated, the affinity, specificity, and nature of which may promote, inhibit, or define the course of assembly. Despite the importance of the intermolecular interactions that initiate amyloid assembly, our understanding of these events remains poor. Here, using amyloidogenic and nonamyloidogenic variants of β2-microglobulin, we identify the interactions that inhibit or promote fibril formation in atomic detail. The results reveal that different outcomes of assembly result from biomolecular interactions involving similar surfaces. Specifically, inhibition occurs via rigid body docking of monomers in a head-to-head orientation to form kinetically trapped dimers. By contrast, the promotion of fibrillation involves relatively weak protein association in a similar orientation, which results in conformational changes in the initially nonfibrillogenic partner. The results highlight the complexit...
RATIONALE: Amyloid formation is implicated in a number of human diseases. b 2-microglobulin (b 2 ... more RATIONALE: Amyloid formation is implicated in a number of human diseases. b 2-microglobulin (b 2 m) is the precursor protein in dialysis-related amyloidosis and it has been shown that partial, or more complete, unfolding is key to amyloid fibril formation in this pathology. Here the relationship between conformational flexibility and b 2 m amyloid formation at physiological pH has been investigated. METHODS: HDX-ESI-MS was used to study the conformational dynamics of b 2 m. Protein engineering, or the addition of Cu 2+ ions, sodium dodecyl sulphate, trifluoroethanol, heparin, or protein stabilisers, was employed to perturb the conformational dynamics of b 2 m. The fibril-forming propensities of the protein variants and the wild-type protein in the presence of additives, which resulted in >5-fold increase in the EX1 rate of HDX, were investigated further. RESULTS: ESI-MS revealed that HDX occurs via a mixed EX1/EX2 mechanism under all conditions. Urea denaturation and tryptophan fluorescence indicated that EX1 exchange occurred from a globally unfolded state in wild-type b 2 m. Although >30-fold increase in the HDX exchange rate was observed both for the protein variants and for the wild-type protein in the presence of specific additives, large increases in exchange rate did not necessarily result in extensive de novo fibril formation. CONCLUSIONS: The conformational dynamics measured by the EX1 rate of HDX do not predict the ability of b 2 m to form amyloid fibrils de novo at neutral pH. This suggests that the formation of amyloid fibrils from b 2 m at neutral pH is dependent on the generation of one or more specific aggregation-competent species which facilitate self-assembly.
Proceedings of the National Academy of Sciences, 2010
The key to understanding amyloid disease is the characterization of oligomeric species formed dur... more 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...
Proceedings of the National Academy of Sciences, 2007
Nanoparticles present enormous surface areas and are found to enhance the rate of protein fibrill... more 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 β 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 β 2 -microglobulin forms multiple layers on the particle surface, providing a locally increased protein concentration pro...
Proceedings of the National Academy of Sciences, 2012
Protein misfolding and aggregation cause serious degenerative conditions such as Alzheimer’s, Par... more 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 β 2 -microglobulin (β 2 m). Using cryoelectron tomography, we demonstrate that fragmented β 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 poin...
Although small molecules that modulate amyloid formation in vitro have been identified, significa... more Although small molecules that modulate amyloid formation in vitro have been identified, significant challenges remain in determining precisely how these species act. Here we describe the identification of rifamycin SV as a potent inhibitor of β 2 m fibrillogenesis when added during the lag time of assembly or early during fibril elongation. Biochemical experiments demonstrate that the small molecule does not act by a colloidal mechanism. Exploiting the ability of electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) to resolve intermediates of amyloid assembly, we show instead that rifamycin SV inhibits β 2 m fibrillation by binding distinct monomeric conformers, disfavoring oligomer formation, and diverting the course of assembly to the formation of spherical aggregates. The results reveal the power of ESI-IMS-MS to identify specific protein conformers as targets for intervention in fibrillogenesis using small molecules and reveal a mechanism of action in which ligand binding diverts unfolded protein monomers towards alternative assembly pathways. Keywords β 2-microglobulin; amyloid; rifamycin SV; oligomers; NMR; ESI-MS Aberrant aggregation of proteins into amyloid fibrils is a characteristic of over twenty-five human disorders. 1 In each case, the precursor protein is dissimilar in terms of its native fold and primary sequence, yet amyloid fibrils share common structural and tinctorial features, such as a cross-β organization of the polypeptide chain and the ability to bind dyes such as thioflavin-T (ThT) and Congo red. 2 The observation of a common structural architecture for amyloid fibrils has motivated efforts to elucidate the molecular mechanisms of fibril formation in vitro with the aim of revealing possible targets for therapeutic intervention. Such investigations have led to the observation of different species populated en route to amyloid fibrils, including oligomers of different size and morphology, as well as protofibrils, annular aggregates and worm-like assemblies. 3-6 The heterogeneity, dynamic
Characterising the differences between oligomers formed from the amyloidogenic protein β2-microgl... more Characterising the differences between oligomers formed from the amyloidogenic protein β2-microglobulin and its mutant H51A using ESI-IMS-MS.
Amyloid fibrils are ordered polymers in which constituent polypeptides adopt a non-native fold. D... more Amyloid fibrils are ordered polymers in which constituent polypeptides adopt a non-native fold. Despite their importance in degenerative human diseases, the overall structure of amyloid fibrils remains unknown. High-resolution studies of model peptide assemblies have identified residues forming cross-β-strands and have revealed some details of local β-strand packing. However, little is known about the assembly contacts that define the fibril architecture. Here we present a set of threedimensional structures of amyloid fibrils formed from full-length β 2-microglobulin, a 99-residue protein involved in clinical amyloidosis. Our cryo-electron microscopy maps reveal a hierarchical fibril structure built from tetrameric units of globular density, with at least three different subunit interfaces in this homopolymeric assembly. These findings suggest a more complex superstructure for amyloid than hitherto suspected and prompt a re-evaluation of the defining features of the amyloid fold. Abbreviations FTIR, Fourier transform infrared; 3D, three-dimensional; EM, electron microscopy; β 2 m, β 2microglobulin; STEM, scanning transmission electron microscopy; MPL, mass per unit length
Journal of the American Chemical Society, May 18, 2016
The balance between protein folding and misfolding is a crucial determinant of amyloid assembly. ... more The balance between protein folding and misfolding is a crucial determinant of amyloid assembly. Transient intermediates that are sparsely populated during protein folding have been identified as key players in amyloid aggregation. However, due to their ephemeral nature, structural characterization of these species remains challenging. Here, using the power of nonuniformly sampled NMR methods we investigate the folding pathway of amyloidogenic and nonamyloidogenic variants of β2-microglobulin (β2m) in atomic detail. Despite folding via common intermediate states, we show that the decreased population of the aggregation-prone ITrans state and population of a less stable, more dynamic species ablate amyloid formation by increasing the energy barrier for amyloid assembly. The results show that subtle changes in conformational dynamics can have a dramatic effect in determining whether a protein is amyloidogenic, without perturbation of the mechanism of protein folding.
Proceedings of the National Academy of Sciences of the United States of America, Jan 20, 2015
Amyloid disorders cause debilitating illnesses through the formation of toxic protein aggregates.... more Amyloid disorders cause debilitating illnesses through the formation of toxic protein aggregates. The mechanisms of amyloid toxicity and the nature of species responsible for mediating cellular dysfunction remain unclear. Here, using β2-microglobulin (β2m) as a model system, we show that the disruption of membranes by amyloid fibrils is caused by the molecular shedding of membrane-active oligomers in a process that is dependent on pH. Using thioflavin T (ThT) fluorescence, NMR, EM and fluorescence correlation spectroscopy (FCS), we show that fibril disassembly at pH 6.4 results in the formation of nonnative spherical oligomers that disrupt synthetic membranes. By contrast, fibril dissociation at pH 7.4 results in the formation of nontoxic, native monomers. Chemical cross-linking or interaction with hsp70 increases the kinetic stability of fibrils and decreases their capacity to cause membrane disruption and cellular dysfunction. The results demonstrate how pH can modulate the delete...
In the early stages of amyloid formation, heterogeneous populations of oligomeric species are gen... more In the early stages of amyloid formation, heterogeneous populations of oligomeric species are generated, the affinity, specificity, and nature of which may promote, inhibit, or define the course of assembly. Despite the importance of the intermolecular interactions that initiate amyloid assembly, our understanding of these events remains poor. Here, using amyloidogenic and nonamyloidogenic variants of β2-microglobulin, we identify the interactions that inhibit or promote fibril formation in atomic detail. The results reveal that different outcomes of assembly result from biomolecular interactions involving similar surfaces. Specifically, inhibition occurs via rigid body docking of monomers in a head-to-head orientation to form kinetically trapped dimers. By contrast, the promotion of fibrillation involves relatively weak protein association in a similar orientation, which results in conformational changes in the initially nonfibrillogenic partner. The results highlight the complexit...
RATIONALE: Amyloid formation is implicated in a number of human diseases. b 2-microglobulin (b 2 ... more RATIONALE: Amyloid formation is implicated in a number of human diseases. b 2-microglobulin (b 2 m) is the precursor protein in dialysis-related amyloidosis and it has been shown that partial, or more complete, unfolding is key to amyloid fibril formation in this pathology. Here the relationship between conformational flexibility and b 2 m amyloid formation at physiological pH has been investigated. METHODS: HDX-ESI-MS was used to study the conformational dynamics of b 2 m. Protein engineering, or the addition of Cu 2+ ions, sodium dodecyl sulphate, trifluoroethanol, heparin, or protein stabilisers, was employed to perturb the conformational dynamics of b 2 m. The fibril-forming propensities of the protein variants and the wild-type protein in the presence of additives, which resulted in >5-fold increase in the EX1 rate of HDX, were investigated further. RESULTS: ESI-MS revealed that HDX occurs via a mixed EX1/EX2 mechanism under all conditions. Urea denaturation and tryptophan fluorescence indicated that EX1 exchange occurred from a globally unfolded state in wild-type b 2 m. Although >30-fold increase in the HDX exchange rate was observed both for the protein variants and for the wild-type protein in the presence of specific additives, large increases in exchange rate did not necessarily result in extensive de novo fibril formation. CONCLUSIONS: The conformational dynamics measured by the EX1 rate of HDX do not predict the ability of b 2 m to form amyloid fibrils de novo at neutral pH. This suggests that the formation of amyloid fibrils from b 2 m at neutral pH is dependent on the generation of one or more specific aggregation-competent species which facilitate self-assembly.
Proceedings of the National Academy of Sciences, 2010
The key to understanding amyloid disease is the characterization of oligomeric species formed dur... more 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...
Proceedings of the National Academy of Sciences, 2007
Nanoparticles present enormous surface areas and are found to enhance the rate of protein fibrill... more 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 β 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 β 2 -microglobulin forms multiple layers on the particle surface, providing a locally increased protein concentration pro...
Proceedings of the National Academy of Sciences, 2012
Protein misfolding and aggregation cause serious degenerative conditions such as Alzheimer’s, Par... more 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 β 2 -microglobulin (β 2 m). Using cryoelectron tomography, we demonstrate that fragmented β 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 poin...
Although small molecules that modulate amyloid formation in vitro have been identified, significa... more Although small molecules that modulate amyloid formation in vitro have been identified, significant challenges remain in determining precisely how these species act. Here we describe the identification of rifamycin SV as a potent inhibitor of β 2 m fibrillogenesis when added during the lag time of assembly or early during fibril elongation. Biochemical experiments demonstrate that the small molecule does not act by a colloidal mechanism. Exploiting the ability of electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) to resolve intermediates of amyloid assembly, we show instead that rifamycin SV inhibits β 2 m fibrillation by binding distinct monomeric conformers, disfavoring oligomer formation, and diverting the course of assembly to the formation of spherical aggregates. The results reveal the power of ESI-IMS-MS to identify specific protein conformers as targets for intervention in fibrillogenesis using small molecules and reveal a mechanism of action in which ligand binding diverts unfolded protein monomers towards alternative assembly pathways. Keywords β 2-microglobulin; amyloid; rifamycin SV; oligomers; NMR; ESI-MS Aberrant aggregation of proteins into amyloid fibrils is a characteristic of over twenty-five human disorders. 1 In each case, the precursor protein is dissimilar in terms of its native fold and primary sequence, yet amyloid fibrils share common structural and tinctorial features, such as a cross-β organization of the polypeptide chain and the ability to bind dyes such as thioflavin-T (ThT) and Congo red. 2 The observation of a common structural architecture for amyloid fibrils has motivated efforts to elucidate the molecular mechanisms of fibril formation in vitro with the aim of revealing possible targets for therapeutic intervention. Such investigations have led to the observation of different species populated en route to amyloid fibrils, including oligomers of different size and morphology, as well as protofibrils, annular aggregates and worm-like assemblies. 3-6 The heterogeneity, dynamic
Characterising the differences between oligomers formed from the amyloidogenic protein β2-microgl... more Characterising the differences between oligomers formed from the amyloidogenic protein β2-microglobulin and its mutant H51A using ESI-IMS-MS.
Amyloid fibrils are ordered polymers in which constituent polypeptides adopt a non-native fold. D... more Amyloid fibrils are ordered polymers in which constituent polypeptides adopt a non-native fold. Despite their importance in degenerative human diseases, the overall structure of amyloid fibrils remains unknown. High-resolution studies of model peptide assemblies have identified residues forming cross-β-strands and have revealed some details of local β-strand packing. However, little is known about the assembly contacts that define the fibril architecture. Here we present a set of threedimensional structures of amyloid fibrils formed from full-length β 2-microglobulin, a 99-residue protein involved in clinical amyloidosis. Our cryo-electron microscopy maps reveal a hierarchical fibril structure built from tetrameric units of globular density, with at least three different subunit interfaces in this homopolymeric assembly. These findings suggest a more complex superstructure for amyloid than hitherto suspected and prompt a re-evaluation of the defining features of the amyloid fold. Abbreviations FTIR, Fourier transform infrared; 3D, three-dimensional; EM, electron microscopy; β 2 m, β 2microglobulin; STEM, scanning transmission electron microscopy; MPL, mass per unit length
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Papers by Sheena Radford