Mapping the core of the β2-microglobulin amyloid fibril by H/D exchange

Hoshino, Minoru; Katou, Hidenori; Hagihara, Yoshihisa; Hara, Kazuhiro; Naiki, Hironobu; Goto, Yuji

doi:10.1038/nsb792

Cited by 328 publications

(406 citation statements)

References 31 publications

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“…Importantly, dissolution of the amyloid fibrils by 4 M guanidinium thiocyanate in 50% H 2 O/D 2 O enabled observation of almost all residues (Figure 2), including residues which are part of the disulfide-bridged strands B and F in the native structure that were not detected in previous H/D exchange experiments. 20 Our data show that parts of strands B and F stay protected from solvent exchange and are buried in the fibrillar core. Importantly, reduction of the disulfide bridge leads to formation of fibrils with different morphology.…”

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confidence: 61%

Molten Globule Precursor States Are Conformationally Correlated to Amyloid Fibrils of Human β-2-Microglobulin

2010

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Misfolding intermediates play a key role in defining aberrant protein aggregation and amyloid formation in more than 15 different human diseases. However, their experimental characterization is challenging due to the transient nature and conformational heterogeneity of the involved states. Here, we demonstrate that direct carbon-detected NMR experiments allow observation, assignment, and structural analysis of molten globule amyloid intermediates that are severely broadened by conformational exchange. The method is used to characterize the structure and dynamics of partially unfolded intermediates of the 99-residue protein -2-microglobulin, which is the major component of insoluble aggregates occurring in dialysis-related amyloidosis. Comparison of the conformational properties of the molten globule-like intermediates with levels of deuterium incorporation into amyloid fibrils of -2-microglobulin revealed a close relationship between the conformational properties of the metastable intermediates and the -sheet-rich insoluble aggregates of -2-microglobulin.Misfolding intermediates play a key role in defining aberrant protein aggregation and amyloid formation in more than 15 different human diseases. 1,2 However, the experimental characterization of the conformation of amyloid intermediates is challenging due to their transient nature and conformational heterogeneity. 3,4 This severely limits our knowledge about the relation of the conformation of the precursor states to the structure of amyloid fibrils.Dialysis-related amyloidosis is a protein misfolding disease resulting from deposition of amyloid aggregates in skeletal tissue that contain fibrils of the 99-residue-long protein -2-microglobulin (b2m). 5 Amyloid formation of b2m is strongly enhanced in conditions that destabilize its globular structure. 6 This can be achieved in Vitro by acid denaturation, with two distinct intermediate states being formed under acidic conditions. The highest population of the partially unfolded intermediate occurs at pH 3.6, where also the rate of fibril formation reaches a maximum. 7 However, long and straight amyloid fibrils resembling those extracted from patients are formed from a precursor state formed at pH 2.5. 8,9 Here, we compare the conformational properties of these two partially unfolded intermediates with single-residue resolution and demonstrate that a close relationship exists between the structure and dynamics of the amyloid precursor species and the morphology of mature fibrils of b2m.At pH 2.5 b2m is highly unfolded, the majority of resonances are observed in 1 H-15 N HSQC spectra (Supporting Information, Figure 1) and can be assigned to individual residues. 10 In contrast, only about 40-60 resolved peaks on top of a broad hump of unresolved signal intensity (visible at lower contour levels) could be observed at pH 3.6 ( Figure 1a). 11 To obtain sequence-specific information about the pH 3.6 intermediate, we tested direct 13 C detection. 12-14 Direct carbon detection was previously successfully used fo...

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confidence: 61%

Molten Globule Precursor States Are Conformationally Correlated to Amyloid Fibrils of Human β-2-Microglobulin

2010

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“…Similarly, ␤ 2 -microglobulin forms amyloid fibrils under conditions in which the N-and C-terminal ␤-strands of the protein are unfolded (6). Observations that these regions of the sequence are solvent-exposed in the fibrils formed from this protein provide direct evidence that fibril formation does not originate from the assembly of protein molecules in their native conformation (7). Some other proteins such as cystatin C, which is associated with a hereditary form of cerebral amyloid angiopathy, and the cell cycle regulatory protein p13suc1 are suggested to fibrillize via domain swapping (8,9).…”

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confidence: 78%

Aggregation of the Acylphosphatase from Sulfolobus solfataricus

Plakoutsi

Taddei

Stefani

et al. 2004

Journal of Biological Chemistry

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Protein aggregation is associated with a number of human pathologies including Alzheimer's and Creutzfeldt-Jakob diseases and the systemic amyloidoses. In this study, we used the acylphosphatase from the hyperthermophilic Archaea Sulfolobus solfataricus (Sso AcP) to investigate the mechanism of aggregation under conditions in which the protein maintains a folded structure. In the presence of 15-25% (v/v) trifluoroethanol, Sso AcP was found to form aggregates able to bind specific dyes such as thioflavine T, Congo red, and 1-anilino-8-naphthalenesulfonic acid. The presence of aggregates was confirmed by circular dichroism and dynamic light scattering. Electron microscopy revealed the presence of small aggregates generally referred to as amyloid protofibrils. The monomeric form adopted by Sso AcP prior to aggregation under these conditions retained enzymatic activity; in addition, folding was remarkably faster than unfolding. These observations indicate that Sso AcP adopts a folded, although possibly distorted, conformation prior to aggregation. Most important, aggregation appeared to be 100-fold faster than unfolding under these conditions. Although aggregation of Sso AcP was faster at higher trifluoroethanol concentrations, in which the protein adopted a partially unfolded conformation, these findings suggest that the early events of amyloid fibril formation may involve an aggregation process consisting of the assembly of protein molecules in their folded state. This conclusion has a biological relevance as globular proteins normally spend most of their lifetime in folded structures.

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“…The determination of detailed structures of the mature fibrils is also challenging due to their intractable and frequently heterogeneous nature, which again seriously limits the application of the traditional methods of structural biology such as solution NMR spectroscopy and X-ray diffraction techniques. Structural information concerning amyloid fibrils has, however, been obtained from atomic force microscopy (AFM) [30,31], FTIR [32,33], X-ray fibre diffraction studies [17], cryoelectron microscopy [34,35], hydrogen/deuterium exchange analysed by mass spectrometry and NMR [36][37][38][39] and solid state NMR [40,41]. Important information about both the structures of the aggregates and the mechanism of their formation has also been obtained by using methods such as limited proteolysis [42,43], systematic site-directed mutagenesis [44][45][46], and the analysis of the effects of interactions with specific antibodies.…”

Section: The Generic Nature Of the Amyloid Structurementioning

confidence: 99%

Probing the origins, diagnosis and treatment of amyloid diseases using antibodies

Dumoulin

Dobson

2004

Biochimie

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The deposition of proteins in the form of amyloid fibrils is the characteristic feature of more than 20 medical conditions affecting the central nervous system or a variety of peripheral tissues. These disorders, which include Alzheimer's disease, the prion diseases and type II diabetes, are of enormous importance in the context of present-day human health and welfare. Extensive research is therefore being carried out to define the molecular details of the mechanism of the pathological conversion of amyloidogenic proteins from their soluble forms into fibrillar structures. This review focuses on recent studies that demonstrate the power of using antibodies or antibody fragments to probe the process of fibril formation, and discusses the emerging potential of these species as diagnostic and therapeutic agents.

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Mapping the core of the β2-microglobulin amyloid fibril by H/D exchange

Cited by 328 publications

References 31 publications

Molten Globule Precursor States Are Conformationally Correlated to Amyloid Fibrils of Human β-2-Microglobulin

Molten Globule Precursor States Are Conformationally Correlated to Amyloid Fibrils of Human β-2-Microglobulin

Aggregation of the Acylphosphatase from Sulfolobus solfataricus

Probing the origins, diagnosis and treatment of amyloid diseases using antibodies

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