Dynamics in the Unfolded State of β2-microglobulin Studied by NMR (original) (raw)
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The role of conformational flexibility in β 2 ‐microglobulin amyloid fibril formation at neutral pH
Rapid Communications in Mass Spectrometry, 2012
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.
Ligand binding to distinct states diverts aggregation of an amyloid-forming protein
Nature Chemical Biology, 2011
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
Biochimica Et Biophysica Acta-proteins and Proteomics, 2005
Deriving a complete understanding of protein self-association into amyloid fibrils across multiple distance and time scales is an enormous challenge. At small length scales, a detailed description of the partially folded protein ensemble that participates in self-assembly remains obscure. At larger length scales, amyloid fibrils are often heterogeneous, can form along multiple pathways, and are further complicated by phenomena such as phase-separation. Over the last 5 years, we have used an array of biophysical approaches in order to elucidate the structural and molecular mechanism of amyloid fibril formation, focusing on the all h-sheet protein, h 2 -microglubulin (h 2 m). This protein forms amyloid deposits in the human disease Fdialysis-related amyloidosis_ (DRA). We have shown that under acidic conditions h 2 m rapidly associates in vitro to form amyloid-like fibrils that have different morphological properties, but which contain an underpinning cross-h structure. In this review, we discuss our current knowledge of the structure of these fibrils, as well as the structural, kinetic and thermodynamic relationship between fibrils with different morphologies. The results provide some of the first insights into the shape of the self-assembly free-energy landscape for this protein and highlight the parallel nature of the assembly process. We include a detailed description of the structure and dynamics of partially folded and acid unfolded species of h 2 m using NMR, and highlight regions thought to be important in early self-association events. Finally, we discuss briefly how knowledge of assembly mechanisms in vitro can be used to inform the design of therapeutic strategies for this, and other amyloid disorders, and we speculate on how the increasing power of biophysical approaches may lead to a fuller description of protein self-assembly into amyloid in the future. D
Conformational Conversion during Amyloid Formation at Atomic Resolution
Molecular Cell, 2011
Numerous studies of amyloid assembly have indicated that partially folded protein species are responsible for initiating aggregation. Despite their importance, the structural and dynamic features of amyloidogenic intermediates and the molecular details of how they cause aggregation remain elusive. Here, we use ΔN6, a truncation variant of the naturally amyloidogenic protein β 2microglobulin (β 2 m), to determine the solution structure of a nonnative amyloidogenic intermediate at high resolution. The structure of ΔN6 reveals a major repacking of the hydrophobic core to accommodate the nonnative peptidyl-prolyl trans-isomer at Pro32. These structural changes, together with a concomitant pH-dependent enhancement in backbone dynamics on a microsecond-millisecond timescale, give rise to a rare conformer with increased amyloidogenic potential. We further reveal that catalytic amounts of ΔN6 are competent to convert nonamyloidogenic human wild-type β 2 m (Hβ 2 m) into a rare amyloidogenic conformation and provide structural evidence for the mechanism by which this conformational conversion occurs.
Amyloid formation under physiological conditions proceeds via a native-like folding intermediate
Nature Structural & Molecular Biology, 2006
Although most proteins can assemble into amyloid-like fibrils in vitro under extreme conditions, how proteins form amyloid fibrils in vivo remains unresolved. Identifying rare aggregation-prone species under physiologically relevant conditions and defining their structural properties is therefore an important challenge. By solving the folding mechanism of the naturally amyloidogenic protein beta-2-microglobulin at pH 7.0 and 37 degrees C and correlating the concentrations of different species with the rate of fibril elongation, we identify a specific folding intermediate, containing a non-native trans-proline isomer, as the direct precursor of fibril elongation. Structural analysis using NMR shows that this species is highly native-like but contains perturbation of the edge strands that normally protect beta-sandwich proteins from self-association. The results demonstrate that aggregation pathways can involve self-assembly of highly native-like folding intermediates, and have implications for the prevention of this, and other, amyloid disorders.
Proceedings of the National Academy of Sciences, 2013
The self-assembly of abnormally folded proteins into amyloid fibrils is a hallmark of many debilitating diseases, from Alzheimer's and Parkinson diseases to prion-related disorders and diabetes type II. However, the fundamental mechanism of amyloid aggregation remains poorly understood. Core sequences of four to seven amino acids within natural amyloid proteins that form toxic fibrils have been used to study amyloidogenesis. We recently reported a class of systematically designed ultrasmall peptides that self-assemble in water into cross-β-type fibers. Here we compare the self-assembly of these peptides with natural core sequences. These include core segments from Alzheimer's amyloid-β, human amylin, and calcitonin. We analyzed the self-assembly process using circular dichroism, electron microscopy, X-ray diffraction, rheology, and molecular dynamics simulations. We found that the designed aliphatic peptides exhibited a similar self-assembly mechanism to several natural sequences, with formation of α-helical intermediates being a common feature. Interestingly, the self-assembly of a second core sequence from amyloid-β, containing the diphenylalanine motif, was distinctly different from all other examined sequences. The diphenylalanine-containing sequence formed β-sheet aggregates without going through the α-helical intermediate step, giving a unique fiberdiffraction pattern and simulation structure. Based on these results, we propose a simplified aliphatic model system to study amyloidosis. Our results provide vital insight into the nature of early intermediates formed and suggest that aromatic interactions are not as important in amyloid formation as previously postulated. This information is necessary for developing therapeutic drugs that inhibit and control amyloid formation.
A Population Shift between Sparsely Populated Folding Intermediates Determines Amyloidogenicity
Journal of the American Chemical Society, 2016
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.
Structural mapping of oligomeric intermediates in an amyloid assembly pathway
eLife
Transient oligomers are commonly formed in the early stages of amyloid assembly. Determining the structure(s) of these species and defining their role(s) in assembly is key to devising new routes to control disease. Here, using a combination of chemical kinetics, NMR spectroscopy and other biophysical methods, we identify and structurally characterize the oligomers required for amyloid assembly of the protein DN6, a truncation variant of human β2-microglobulin (b2m) found in amyloid deposits in the joints of patients with dialysis-related amyloidosis. The results reveal an assembly pathway which is initiated by the formation of head-to-head non-toxic dimers and hexamers en route to amyloid fibrils. Comparison with inhibitory dimers shows that precise subunit organization determines amyloid assembly, while dynamics in the C-terminal strand hint to the initiation of cross-β structure formation. The results provide a detailed structural view of early amyloid assembly involving structur...
Competition between intramolecular and intermolecular interactions in an amyloid-forming protein
Journal of molecular biology, 2009
Despite much progress in understanding the folding and the aggregation processes of proteins, the rules defining their interplay have yet to be fully defined. This problem is of particular importance since many diseases are initiated by protein unfolding and hence the propensity to aggregate competes with intramolecular collapse and other folding events. Here, we describe the roles of intramolecular and intermolecular interactions in defining the length of the lag time and the apparent rate of elongation of the 100-residue protein human beta(2)-microglobulin at pH 2.5, commencing from an acid-denatured state that lacks persistent structure but contains significant non-random hydrophobic interactions. Using a combination of site-directed mutagenesis, quantitative kinetic analysis and computational methods, we show that only a single region of about 10 residues in length, determines the rate of fibril formation, despite the fact that other regions exhibit a significant intrinsic prope...