Structural properties of an amyloid precursor of β2-microglobulin

McParland, Victoria; Kalverda, Arnout P.; Homans, Steve W.; Radford, Sheena E.

doi:10.1038/nsb791

Cited by 191 publications

(188 citation statements)

References 30 publications

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“…Assignment of Urea-denatured State-To determine the structural characteristics of ␤-PrP on a per residue basis, it is necessary to recover the line-broadened signals, a task that is often readily achieved through the addition of a chemical denaturant (50,60). Previous experiments demonstrated that ␤-PrP is prone to aggregation at high ionic strength and therefore urea was used here as an alternative to guanidine hydrochloride, to minimize any aggregation (58).…”

Section: Resultsmentioning

confidence: 99%

Conformational Properties of β-PrP

Hosszu

Trevitt

Jones

et al. 2009

Journal of Biological Chemistry

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Prion propagation involves a conformational transition of the cellular form of prion protein (PrP C ) to a disease-specific isomer (PrP Sc ), shifting from a predominantly ␣-helical conformation to one dominated by ␤-sheet structure. This conformational transition is of critical importance in understanding the molecular basis for prion disease. Here, we elucidate the conformational properties of a disulfide-reduced fragment of human PrP spanning residues 91-231 under acidic conditions, using a combination of heteronuclear NMR, analytical ultracentrifugation, and circular dichroism. We find that this form of the protein, which similarly to PrP Sc , is a potent inhibitor of the 26 S proteasome, assembles into soluble oligomers that have significant ␤-sheet content. The monomeric precursor to these oligomers exhibits many of the characteristics of a molten globule intermediate with some helical character in regions that form helices I and III in the PrP C conformation, whereas helix II exhibits little evidence for adopting a helical conformation, suggesting that this region is a likely source of interaction within the initial phases of the transformation to a ␤-rich conformation. This precursor state is almost as compact as the folded PrP C structure and, as it assembles, only residues 126 -227 are immobilized within the oligomeric structure, leaving the remainder in a mobile, random-coil state.

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Section: Resultsmentioning

confidence: 99%

Conformational Properties of β-PrP

Hosszu

Trevitt

Jones

et al. 2009

Journal of Biological Chemistry

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“…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][13][14] Direct carbon detection was previously successfully used for the study of unfolded proteins and to alleviate problems of exchange.…”

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confidence: 99%

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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“…This is consistent with the findings from other previous reports -an acetate buffer solution at pH 5.5 with trifluoroethanol was used to induce soluble oligomers of SH3 domain from bovine phosphatidyl-inositol-3'-kinase and the amino-terminal domain of the Escherichia coli HypF protein 21 . Human β 2 -microglobulin and transthyretin form amyloid fibrils efficiently under acidic conditions in vitro 22,24,25 . For functional amyloidogenic protein Pme17, a critical pH regime between 5 +/-0.5 is required for fibril formation in a specific melanosome compartment 26 .…”

Section: Discussionmentioning

confidence: 99%

“…This is dictated by the high protein concentration necessary to permit the effective aggregation of partially misfolded proteins in order to form soluble protein oligomers. Previously, protein-only amyloid has been generated by treating native proteins with high temperature, hydrophobic solvent, denaturing agents, or extreme pH exposure [19][20][21][22] . However, soluble protein oligomers, if detectable at all, are only formed temporarily in solution with very limited abundance.…”

Section: Discussionmentioning

confidence: 99%

“…Previously, various methods have been developed to generate amyloid in vitro by applying conditions known to favor protein misfolding, such as high temperature, hydrophobic environment, and pH variations [19][20][21][22][23] . However, they associate with various problems, such as low efficiency, reversible folding, large batch variations or slow kinetics.…”

Section: Introductionmentioning

confidence: 99%

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Rapid Generation of Amyloid from Native Proteins <em>In vitro</em>

Dorta‐Estremera

Cao

2013

JoVE

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Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion. Video LinkThe video component of this article can be found at

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Structural properties of an amyloid precursor of β2-microglobulin

Cited by 191 publications

References 30 publications

Conformational Properties of β-PrP

Conformational Properties of β-PrP

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

Rapid Generation of Amyloid from Native Proteins <em>In vitro</em>

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