Characterization of the Formation of Amyloid Protofibrils from Barstar by Mapping Residue-specific Fluorescence Dynamics

Mukhopadhyay, Samrat; Nayak, Pabitra K.; Udgaonkar, Jayant B.; Krishnamoorthy, G.

doi:10.1016/j.jmb.2006.02.006

Cited by 62 publications

(80 citation statements)

References 34 publications

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“…This helped us to determine the size of the peptide. Rotational anisotropy measurements of the native monomeric state of a model protein barstar (a compact quasispherical protein with a molecular mass of 10 kDa) labeled with EDANS at the C terminus yielded a rotational correlation time of around 3.5 ns (33). We used this as a calibrant.…”

Section: Discussionmentioning

confidence: 99%

Nature of the Amyloid-β Monomer and the Monomer-Oligomer Equilibrium

Nag

Sarkar

Banerjee

et al. 2011

Journal of Biological Chemistry

168

166

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The monomer to oligomer transition initiates the aggregation and pathogenic transformation of Alzheimer amyloid-␤ (A␤) peptide. However, the monomeric state of this aggregationprone peptide has remained beyond the reach of most experimental techniques, and a quantitative understanding of this transition is yet to emerge. Here, we employ single-molecule level fluorescence tools to characterize the monomeric state and the monomer-oligomer transition at physiological concentrations in buffers mimicking the cerebrospinal fluid (CSF). Our measurements show that the monomer has a hydrodynamic radius of 0.9 ؎ 0.1 nm, which confirms the prediction made by some of the in silico studies. Surprisingly, at equilibrium, both A␤ 40 and A␤ 42 remain predominantly monomeric up to 3 M, above which it forms large aggregates. This concentration is much higher than the estimated concentrations in the CSF of either normal or diseased brains. If A␤ oligomers are present in the CSF and are the key agents in Alzheimer pathology, as is generally believed, then these must be released in the CSF as preformed entities. Although the oligomers are thermodynamically unstable, we find that a large kinetic barrier, which is mostly entropic in origin, strongly impedes their dissociation. Thermodynamic principles therefore allow the development of a pharmacological agent that can catalytically convert metastable oligomers into nontoxic monomers.Alzheimer disease (AD) 2 is a degenerative brain disorder that is associated with the presence of extracellular aggregates of amyloid-␤ (A␤) (1), which is an ϳ4.5-kDa peptide containing 39 -42 residues. Recent studies indicate that small soluble oligomers are key to A␤ toxicity (2-4). In the AD brain, both A␤ monomers and dimers have been isolated, and the dimers have been shown to impair synaptic plasticity in mouse hippocampal slices (5). In contrast, A␤ monomers have been shown to be devoid of neurotoxicity (5) and have in fact been suggested to be neuroprotective (6, 7). The monomer to oligomer transition is therefore not only the obligatory first event of aggregation, it is also the key event determining the transformation of a benign protein to a neurotoxic one.We address this transition from a thermodynamic viewpoint: an aggregation-capable molecule should have a defined equilibrium between monomers and dimers (or oligomers), such that it is primarily monomeric below a certain concentration. Any oligomer-enriched solution prepared below such a concentration must be thermodynamically unstable and must dissociate to monomers at a given rate. To understand AD in terms of A␤ aggregation, we need to understand how this concentration compares with the in vivo concentrations of A␤ (which is estimated to be Ͻ Ͻ1 M) (8 -11) and what the kinetics of A␤ oligomer dissociation is.However, experiments probing the monomer to oligomer transition have been difficult to perform due to the low concentration at which this transition most likely occurs, and they have yielded rather confusing results. Some studies have ...

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Section: Discussionmentioning

confidence: 99%

Nature of the Amyloid-β Monomer and the Monomer-Oligomer Equilibrium

Nag

Sarkar

Banerjee

et al. 2011

Journal of Biological Chemistry

168

166

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“…[54][55][56][57][58][59][60][61] Their exact role in the aggregation process is, however, still controversial. At pH 2, native moPrP is converted into β-rich oligomers and α-rich monomers, which are in slow equilibrium with each other.…”

Section: Discussionmentioning

confidence: 99%

Evidence for Stepwise Formation of Amyloid Fibrils by the Mouse Prion Protein

Jain

Udgaonkar

2008

Journal of Molecular Biology

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120

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“…First, we took advantage of recent developments in single-molecule technology that allow the fluorescence anisotropy of individual molecular species to be determined in real time. The anisotropy of a fluorophore-labeled protein is related to its rotational correlation time, which, in turn, is proportional to the third power of its hydrodynamic radius (19). This provides an extremely sensitive measure of size (20).…”

Section: Probing Oligomeric Nm Intermediates By Single Molecule Fluormentioning

confidence: 99%

Conserved features of intermediates in amyloid assembly determine their benign or toxic states

Krishnan

Goodman

Mukhopadhyay

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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120

127

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Some amyloid-forming polypeptides are associated with devastating human diseases and others provide important biological functions. For both, oligomeric intermediates appear during amyloid assembly. Currently we have few tools for characterizing these conformationally labile intermediates and discerning what governs their benign versus toxic states. Here, we examine intermediates in the assembly of a normal, functional amyloid, the priondetermining region of yeast Sup35 (NM). During assembly, NM formed a variety of oligomers with different sizes and conformation-specific antibody reactivities. Earlier oligomers were less compact and reacted with the conformational antibody A11. More mature oligomers were more compact and reacted with conformational antibody OC. We found we could arrest NM in either of these two distinct oligomeric states with small molecules or crosslinking. The A11-reactive oligomers were more hydrophobic (as measured by Nile Red binding) and were highly toxic to neuronal cells, while OC-reactive oligomers were less hydrophobic and were not toxic. The A11 and OC antibodies were originally raised against oligomers of Aβ, an amyloidogenic peptide implicated in Alzheimer's disease (AD) that is completely unrelated to NM in sequence. Thus, this natural yeast prion samples two conformational states similar to those sampled by Aβ, and when assembly stalls at one of these two states, but not the other, it becomes extremely toxic. Our results have implications for selective pressures operating on the evolution of amyloid folds across a billion years of evolution. Understanding the features that govern such conformational transitions will shed light on human disease and evolution alike.

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Characterization of the Formation of Amyloid Protofibrils from Barstar by Mapping Residue-specific Fluorescence Dynamics

Cited by 62 publications

References 34 publications

Nature of the Amyloid-β Monomer and the Monomer-Oligomer Equilibrium

Nature of the Amyloid-β Monomer and the Monomer-Oligomer Equilibrium

Evidence for Stepwise Formation of Amyloid Fibrils by the Mouse Prion Protein

Conserved features of intermediates in amyloid assembly determine their benign or toxic states

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