Atomic Force Microscopy under Controlled Conditions Reveals Structure of C-Terminal Region of α-Synuclein in Amyloid Fibrils

Sweers, K.K.M.; Werf, Kees O. van der; Bennink, Martin L.; Subramaniam, Vinod

doi:10.1021/nn300863n

Cited by 58 publications

(62 citation statements)

References 49 publications

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“…[66][67][68] In our study, we utilized AFM in a time-dependent manner to visualize the morphologies of early oligomeric species and their subsequent transition to matured fibrillar species (Fig. [66][67][68] In our study, we utilized AFM in a time-dependent manner to visualize the morphologies of early oligomeric species and their subsequent transition to matured fibrillar species (Fig.…”

Section: Nanoscale Morphological Transitionsmentioning

confidence: 99%

Appearance of annular ring-like intermediates during amyloid fibril formation from human serum albumin

Arya

Kumari

Dalal

et al. 2015

Phys. Chem. Chem. Phys.

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The self-assembly of proteins triggered by a conformational switch into highly ordered β-sheet rich amyloid fibrils has captivated burgeoning interest in recent years due to the involvement of amyloids in a variety of human diseases and a diverse range of biological functions. Here, we have investigated the mechanism of fibrillogenesis of human serum albumin (HSA), an all-α-helical protein, using an array of biophysical tools that include steady-state as well as time-resolved fluorescence, circular dichroism and Raman spectroscopy in conjunction with atomic force microscopy (AFM). Investigations into the temporal evolution of nanoscale morphology using AFM revealed the presence of ring-like intermediates that subsequently transformed into worm-like fibrils presumably by a ring-opening mechanism. Additionally, a multitude of morphologically-diverse oligomers were observed on the pathway to amyloid formation. Kinetic analysis using multiple structural probes in-tandem indicated that HSA amyloid assembly is a concerted process encompassing a major structural change that is primarily mediated by hydrophobic interactions between thermally-induced disordered segments originating in various domains. A slower growth kinetics of aggregates suggested that the protein structural reorganization is a prerequisite for fibril formation. Moreover, time-dependent Raman spectroscopic studies of HSA aggregation provided key molecular insights into the conformational transitions occurring within the protein amide backbone and at the residue-specific level. Our data revealed the emergence of conformationally-diverse disulfides as a consequence of structural reorganization and sequestration of tyrosines into the hydrophobic amyloid core comprising antiparallel cross β-sheets.

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Section: Nanoscale Morphological Transitionsmentioning

confidence: 99%

Appearance of annular ring-like intermediates during amyloid fibril formation from human serum albumin

Arya

Kumari

Dalal

et al. 2015

Phys. Chem. Chem. Phys.

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“…Atomic force microscopy images, acquired with tapping mode in air, of (A) typical mature seed fibrils (formed at pH 6.5) and (B) of a low concentration of seed fibrils after prolonged exposure to monomeric α-synuclein, deposited onto mica; see Materials and Methods for details on the preparation of the seed fibrils and SI Appendix, section 4 for a discussion of the influence of the solution conditions on the properties of the seeds. The fibrils (A and B) have an average height of ∼7 nm and exhibit the typical dimensions and twist of mature α-synuclein amyloid fibrils that consist of several protofilaments (19).…”

Section: α-Synuclein Aggregation At Neutral Ph Is Dominated By Fibrilmentioning

confidence: 99%

Solution conditions determine the relative importance of nucleation and growth processes in α-synuclein aggregation

Buell

Galvagnion

Gaspar³

et al. 2014

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

603

867

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The formation of amyloid fibrils by the intrinsically disordered protein α-synuclein is a hallmark of Parkinson disease. To characterize the microscopic steps in the mechanism of aggregation of this protein we have used in vitro aggregation assays in the presence of preformed seed fibrils to determine the molecular rate constant of fibril elongation under a range of different conditions. We show that α-synuclein amyloid fibrils grow by monomer and not oligomer addition and are subject to higher-order assembly processes that decrease their capacity to grow. We also find that at neutral pH under quiescent conditions homogeneous primary nucleation and secondary processes, such as fragmentation and surface-assisted nucleation, which can lead to proliferation of the total number of aggregates, are undetectable. At pH values below 6, however, the rate of secondary nucleation increases dramatically, leading to a completely different balance between the nucleation and growth of aggregates. Thus, at mildly acidic pH values, such as those, for example, that are present in some intracellular locations, including endosomes and lysosomes, multiplication of aggregates is much faster than at normal physiological pH values, largely as a consequence of much more rapid secondary nucleation. These findings provide new insights into possible mechanisms of α-synuclein aggregation and aggregate spreading in the context of Parkinson disease.seeding | prion-like behavior | neurodegenerative disease | kinetic analysis | electrostatic interactions T he conversion of soluble peptide and protein molecules into insoluble amyloid fibrils is of great interest in fields of science ranging from molecular medicine to nanotechnology (1). The formation of amyloid fibrils is a characteristic feature of a substantial number of increasingly common medical disorders, including neurodegenerative conditions such as Alzheimer's and Parkinson diseases (2). Elucidating the fundamental mechanistic steps involved in the conversion from the soluble to the fibrillar forms of the peptides and proteins involved in such disorders is crucial for understanding their origin and proliferation, and hence for exploring in a rational manner new and effective therapeutic strategies through which to combat their onset or progression (3).One general aspect of amyloid diseases is that once the first aggregates are formed it is very difficult to stop or reverse the aggregation process. This implies that aggregation needs to be studied in both the absence and presence of preformed aggregates, commonly known as seeds, to deepen our understanding of the mechanism of the self-assembly process in vivo. It is possible to define from in vitro studies the rate constants for the multiplicity of microscopic steps that increase the number and total mass of the different types of aggregates that are populated during this process. Such an analysis has recently been carried out for the Aβ42 peptide (4) associated with Alzheimer's disease by combining experimental and theoretical methodolo...

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“…From the d STORM images, we inferred an average fibril diameter of 18 ± 2 nm, which shows that the resolution achieved approaches that of techniques such as AFM. 29 …”

Section: Amyloid Growth From Seed Fibrils Is a Bidirectional Processmentioning

confidence: 99%

Direct Observation of Heterogeneous Amyloid Fibril Growth Kinetics via Two-Color Super-Resolution Microscopy

et al. 2013

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The self-assembly of normally soluble proteins into fibrillar amyloid structures is associated with a range of neurodegenerative disorders, such as Parkinson’s and Alzheimer’s diseases. In the present study, we show that specific events in the kinetics of the complex, multistep aggregation process of one such protein, α-synuclein, whose aggregation is a characteristic hallmark of Parkinson’s disease, can be followed at the molecular level using optical super-resolution microscopy. We have explored in particular the elongation of preformed α-synuclein fibrils; using two-color single-molecule localization microscopy we are able to provide conclusive evidence that the elongation proceeds from both ends of the fibril seeds. Furthermore, the technique reveals a large heterogeneity in the growth rates of individual fibrils; some fibrils exhibit no detectable growth, whereas others extend to more than ten times their original length within hours. These large variations in the growth kinetics can be attributed to fibril structural polymorphism. Our technique offers new capabilities in the study of amyloid growth dynamics at the molecular level and is readily translated to the study of the self-assembly of other nanostructures.

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Atomic Force Microscopy under Controlled Conditions Reveals Structure of C-Terminal Region of α-Synuclein in Amyloid Fibrils

Cited by 58 publications

References 49 publications

Appearance of annular ring-like intermediates during amyloid fibril formation from human serum albumin

Appearance of annular ring-like intermediates during amyloid fibril formation from human serum albumin

Solution conditions determine the relative importance of nucleation and growth processes in α-synuclein aggregation

Direct Observation of Heterogeneous Amyloid Fibril Growth Kinetics via Two-Color Super-Resolution Microscopy

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