Characterization of Inter- and Intramolecular Interactions of Amyloid Fibrils by AFM-Based Single-Molecule Force Spectroscopy

Amyloids are fibrous protein aggregates defined by shared specific structural features. Abnormal accumulation of amyloid in organs leads to amyloidosis, which results in various neurodegenerative diseases. Atomic force microscopy (AFM) has proven to be an excellent tool investigating amyloids; it has been extensively utilized to characterize its morphology, assembly process, and mechanical properties. This review summarizes studies which applied AFM to detect the inter- and intramolecular interactions of amyloid fibrils and classified the influencing factors of amyloid’s nanomechanics in detail. The characteristics of amyloid fibrils driven by inter- and intramolecular interactions, including various morphologies of amyloid fibrils, self-assembly process, and the aggregating pathway, are described. Successful examples where AFM provided abundant information about inter- and intramolecular interactions of amyloid fibrils in different environments are presented. Direct force measurement of intra- or intermolecular interactions utilizing an AFM-based tool, single-molecular force spectroscopy (SMFS), is introduced. Some mechanical information such as elasticity, adhesiveness, and strength was obtained by stretching amyloid fibrils. This review helps researchers in understanding the mechanism of amyloidogenesis and exploring the properties of amyloid using AFM techniques.

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Self-Assembly of a DNA-Based Switchable Linker for Single-Molecule Force Spectroscopy

Biophysical Journal ◽

10.1016/j.bpj.2011.11.982 ◽

2012 ◽

Vol 102 (3) ◽

pp. 181a

Author(s):

Ken Halvorsen ◽

Wesley P. Wong

Keyword(s):

Single Molecule ◽

Self Assembly ◽

Force Spectroscopy ◽

Single Molecule Force Spectroscopy

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Identification and nanomechanical characterization of the fundamental single-strand protofilaments of amyloid α-synuclein fibrils

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1721220115 ◽

2018 ◽

Vol 115 (28) ◽

pp. 7230-7235 ◽

Cited By ~ 31

Author(s):

Francesco Simone Ruggeri ◽

Fabrizio Benedetti ◽

Tuomas P. J. Knowles ◽

Hilal A. Lashuel ◽

Sergey Sekatskii ◽

...

Keyword(s):

Single Molecule ◽

Self Assembly ◽

Amyloid Fibrils ◽

Molecular Assembly ◽

Single Strand ◽

Force Microscopy ◽

Atomic Force ◽

Presynaptic Protein ◽

Polypeptide Chains

The formation and spreading of amyloid aggregates from the presynaptic protein α-synuclein in the brain play central roles in the pathogenesis of Parkinson’s disease. Here, we use high-resolution atomic force microscopy to investigate the early oligomerization events of α-synuclein with single monomer angstrom resolution. We identify, visualize, and characterize directly the smallest elementary unit in the hierarchical assembly of amyloid fibrils, termed here single-strand protofilaments. We show that protofilaments form from the direct molecular assembly of unfolded monomeric α-synuclein polypeptide chains. To unravel protofilaments’ internal structure and elastic properties, we manipulated nanomechanically these species by atomic force spectroscopy. The single-molecule scale identification and characterization of the fundamental unit of amyloid assemblies provide insights into early events underlying their formation and shed light on opportunities for therapeutic intervention at the early stages of aberrant protein self-assembly.

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Selectivity of Protein Interactions along the Aggregation Pathway of α-Synuclein

10.1101/2021.01.29.428744 ◽

2021 ◽

Author(s):

André D. G. Leitão ◽

Paulina Rudolffi Soto ◽

Alexandre Chappard ◽

Akshay Bhumkar ◽

Dominic J. B. Hunter ◽

...

Keyword(s):

Single Molecule ◽

Protein Interactions ◽

Self Assembly ◽

Amyloid Fibrils ◽

Lewy Bodies ◽

Coincidence Detection ◽

Protein Protein Interactions ◽

Assembly Pathway ◽

Protein Partners ◽

Molecular Complexity

AbstractThe aggregation of α-SYN follows a cascade of oligomeric, prefibrillar and fibrillar forms, culminating in the formation of Lewy Bodies (LB), the pathological hallmarks of Parkinson’s Disease in neurons. Whilst α-synuclein is a major contributor to LB, these dense accumulations of protein aggregates and tangles of fibrils contain over 70 different proteins. However, the potential for interactions between these proteins and the different aggregated species of α-SYN is largely unknown. We hypothesized that the proteins present in the Lewy Bodies are trapped or pulled into the aggregates in a hierarchical manner, by binding at specific stages of the aggregation of α-SYN.In this study we uncover a map of interactions of a total of 65 proteins, against different species formed by α-SYN. We measured binding to monomeric α-SYN using AlphaScreen, a sensitive nano-bead assay for detection of protein-protein interactions. To access different oligomeric species, we made use of the pathological mutants of α-SYN (A30P, G51D and A53T), which form oligomeric species with distinct properties. Finally, we used bacterially expressed recombinant α-SYN to generate amyloid fibrils and measure interactions with a pool of GFP-tagged potential partners. Binding to oligomers and fibrils was measured by two-color coincidence detection (TCCD) on a single molecule spectroscopy setup. Overall, we demonstrate that LB components are selectively recruited to specific steps in the formation of the LB, explaining their presence in the inclusions. Only a few proteins were found to interact with α-SYN monomers at detectable levels, and only a subset recognizes the oligomeric α-SYN including autophagosomal proteins. We therefore propose a new model for the formation of Lewy Bodies, where selectivity of protein partners at different steps drives the arrangement of these structures, uncovering new ways to modulate aggregation.Significance StatementThe molecular complexity of the Lewy Bodies has been a major hindrance to a bottom-up reconstruction of these inclusions, protein by protein. This work presents an extensive dataset of protein-protein interactions, showing that despite its small size and absence of structure, α-SYN binds to specific partners in the LB, and that there is a clear selectivity of interactions between the different α-SYN species along the self-assembly pathway. We use single-molecule methods to deconvolute number and size of the co-aggregates, to gain detailed information about the mechanisms of interaction. These observations constitute the basis for the elaboration of a global interactome of α-SYN.

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Scanning Tunneling Microscopy Reveals Single-Molecule Insights into the Self-Assembly of Amyloid Fibrils

ACS Nano ◽

10.1021/nn301708d ◽

2012 ◽

Vol 6 (8) ◽

pp. 6882-6889 ◽

Cited By ~ 26

Author(s):

Nataliya Kalashnyk ◽

Jakob T. Nielsen ◽

Erik H. Nielsen ◽

Troels Skrydstrup ◽

Daniel E. Otzen ◽

...

Keyword(s):

Scanning Tunneling Microscopy ◽

Single Molecule ◽

Self Assembly ◽

Amyloid Fibrils ◽

The Self ◽

Scanning Tunneling ◽

Tunneling Microscopy

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Single-Molecule Force Spectroscopy Reveals Self-Assembly Enhanced Surface Binding of Hydrophobins

Chemistry - A European Journal ◽

10.1002/chem.201801730 ◽

2018 ◽

Vol 24 (37) ◽

pp. 9224-9228 ◽

Cited By ~ 5

Author(s):

Bing Li ◽

Xin Wang ◽

Ying Li ◽

Arja Paananen ◽

Géza R. Szilvay ◽

...

Keyword(s):

Single Molecule ◽

Self Assembly ◽

Force Spectroscopy ◽

Surface Binding ◽

Single Molecule Force Spectroscopy ◽

Enhanced Surface

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Single-molecule analysis of the self-assembly process facilitated by host–guest interactions

Chemical Communications ◽

10.1039/c4cc07919a ◽

2015 ◽

Vol 51 (7) ◽

pp. 1202-1205 ◽

Cited By ~ 22

Author(s):

Fu-Na Meng ◽

Xuyang Yao ◽

Yi-Lun Ying ◽

Junji Zhang ◽

He Tian ◽

...

Keyword(s):

Single Molecule ◽

Self Assembly ◽

Methyl Viologen ◽

The Self ◽

Assembly Process ◽

Single Molecule Level ◽

Single Molecule Analysis

The self-assembly process operated by para-sulfonatocalix[6]arenes and methyl viologen was analyzed at the single-molecule level through an α-hemolysin nanopore.

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Fibrous Scaffolds From Elastin-Based Materials

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.652384 ◽

2021 ◽

Vol 9 ◽

Author(s):

Jose Carlos Rodriguez-Cabello ◽

Israel Gonzalez De Torre ◽

Miguel González-Pérez ◽

Fernando González-Pérez ◽

Irene Montequi

Keyword(s):

Self Assembly ◽

Materials Science ◽

Structural Features ◽

Polymer Processing ◽

Current Status ◽

New Paradigm ◽

Processing Technologies ◽

Natural Systems ◽

Fibrous Protein ◽

Manufacturing Technologies

Current cutting-edge strategies in biomaterials science are focused on mimicking the design of natural systems which, over millions of years, have evolved to exhibit extraordinary properties. Based on this premise, one of the most challenging tasks is to imitate the natural extracellular matrix (ECM), due to its ubiquitous character and its crucial role in tissue integrity. The anisotropic fibrillar architecture of the ECM has been reported to have a significant influence on cell behaviour and function. A new paradigm that pivots around the idea of incorporating biomechanical and biomolecular cues into the design of biomaterials and systems for biomedical applications has emerged in recent years. Indeed, current trends in materials science address the development of innovative biomaterials that include the dynamics, biochemistry and structural features of the native ECM. In this context, one of the most actively studied biomaterials for tissue engineering and regenerative medicine applications are nanofiber-based scaffolds. Herein we provide a broad overview of the current status, challenges, manufacturing methods and applications of nanofibers based on elastin-based materials. Starting from an introduction to elastin as an inspiring fibrous protein, as well as to the natural and synthetic elastin-based biomaterials employed to meet the challenge of developing ECM-mimicking nanofibrous-based scaffolds, this review will follow with a description of the leading strategies currently employed in nanofibrous systems production, which in the case of elastin-based materials are mainly focused on supramolecular self-assembly mechanisms and the use of advanced manufacturing technologies. Thus, we will explore the tendency of elastin-based materials to form intrinsic fibers, and the self-assembly mechanisms involved. We will describe the function and self-assembly mechanisms of silk-like motifs, antimicrobial peptides and leucine zippers when incorporated into the backbone of the elastin-based biomaterial. Advanced polymer-processing technologies, such as electrospinning and additive manufacturing, as well as their specific features, will be presented and reviewed for the specific case of elastin-based nanofiber manufacture. Finally, we will present our perspectives and outlook on the current challenges facing the development of nanofibrous ECM-mimicking scaffolds based on elastin and elastin-like biomaterials, as well as future trends in nanofabrication and applications.

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