Visualization of intrinsically disordered proteins by high-speed atomic force microscopy

2022 ◽  
Vol 72 ◽  
pp. 260-266
Author(s):  
Noriyuki Kodera ◽  
Toshio Ando
Keyword(s):  
Atomic Force Microscopy ◽  
High Speed ◽  
Intrinsically Disordered Proteins ◽  
Disordered Proteins ◽  
Force Microscopy ◽  
Intrinsically Disordered ◽  
Atomic Force

scholarly journals Atomic force microscopy reveals structural variability amongst nuclear pore complexes

2018 ◽  
Vol 1 (4) ◽  
pp. e201800142 ◽  
Author(s):  
George J Stanley ◽  
Ariberto Fassati ◽  
Bart W Hoogenboom
Keyword(s):  
Atomic Force Microscopy ◽  
Intrinsically Disordered Proteins ◽  
Structural Diversity ◽  
Disordered Proteins ◽  
Energetic Cost ◽  
Nuclear Pore ◽  
Central Channel ◽  
Force Microscopy ◽  
Intrinsically Disordered ◽  
Atomic Force

The nuclear pore complex (NPC) is a proteinaceous assembly that regulates macromolecular transport into and out of the nucleus. Although the structure of its scaffold is being revealed in increasing detail, its transport functionality depends upon an assembly of intrinsically disordered proteins (called FG-Nups) anchored inside the pore's central channel, which have hitherto eluded structural characterization. Here, using high-resolution atomic force microscopy, we provide a structural and nanomechanical analysis of individual NPCs. Our data highlight the structural diversity and complexity at the nuclear envelope, showing the interplay between the lamina network, actin filaments, and the NPCs. It reveals the dynamic behaviour of NPC scaffolds and displays pores of varying sizes. Of functional importance, the NPC central channel shows large structural diversity, supporting the notion that FG-Nup cohesiveness is in a range that facilitates collective rearrangements at little energetic cost. Finally, different nuclear transport receptors are shown to interact in qualitatively different ways with the FG-Nups, with particularly strong binding of importin-β.

scholarly journals Quantification of biomolecular dynamics inside real and synthetic nuclear pore complexes using time-resolved atomic force microscopy

2019 ◽  
Author(s):  
George J. Stanley ◽  
Bernice Akpinar ◽  
Qi Shen ◽  
Patrick D. Ellis Fisher ◽  
C. Patrick Lusk ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Intrinsically Disordered Proteins ◽  
Stochastic Dynamics ◽  
Disordered Proteins ◽  
Nuclear Pore ◽  
Transport Barrier ◽  
Force Microscopy ◽  
Intrinsically Disordered ◽  
Atomic Force ◽  
Biomolecular Dynamics

AbstractOver the past decades, atomic force microscopy (AFM) has emerged as an increasingly powerful tool to study the dynamics of biomolecules at nanometre length scales. However, the more stochastic the nature of such biomolecular dynamics, the harder it becomes to distinguish them from AFM measurement noise. Rapid, stochastic dynamics are inherent to biological systems comprising intrinsically disordered proteins. One role of such proteins is in the formation of the transport barrier of the nuclear pore complex (NPC): the selective gateway for macromolecular traffic entering or exiting the nucleus. Here, we use AFM to observe the dynamics of intrinsically disordered proteins from two systems: the transport barrier of native NPCs, and the transport barrier of a mimetic NPC made using a DNA origami scaffold. Analysing data recorded with 50-200 ms temporal resolution, we highlight the importance of drift correction and appropriate baseline measurements in such experiments. In addition, we describe an auto-correlation analysis to quantify time scales of observed dynamics and to assess their veracity — an analysis protocol that lends itself to the quantification of stochastic fluctuations in other biomolecular systems. The results reveal the surprisingly slow rate of stochastic, collective transitions inside mimetic NPCs, highlighting the importance of FG-nup cohesive interactions.

scholarly journals Planar Boronic Graphene and Nitrogenized Graphene Heterostructure for Protein Stretch and Confinement

Biomolecules ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1756
Author(s):  
Xuchang Su ◽  
Zhi He ◽  
Lijun Meng ◽  
Hong Liang ◽  
Ruhong Zhou
Keyword(s):  
Single Molecule ◽  
Intrinsically Disordered Proteins ◽  
Electron Tunneling ◽  
Amyloid Β ◽  
Protein Sequencing ◽  
Disordered Proteins ◽  
Force Microscopy ◽  
Intrinsically Disordered ◽  
Atomic Force ◽  
Dynamics Simulations

Single-molecule techniques such as electron tunneling and atomic force microscopy have attracted growing interests in protein sequencing. For these methods, it is critical to refine and stabilize the protein sample to a “suitable mode” before applying a high-fidelity measurement. Here, we show that a planar heterostructure comprising boronic graphene (BC3) and nitrogenized graphene (C3N) sandwiched stripe (BC3/C3N/BC3) is capable of the effective stretching and confinement of three types of intrinsically disordered proteins (IDPs), including amyloid-β (1–42), polyglutamine (Q42), and α-Synuclein (61–95). Our molecular dynamics simulations demonstrate that the protein molecules interact more strongly with the C3N stripe than the BC3 one, which leads to their capture, elongation, and confinement along the center C3N stripe of the heterostructure. The conformational fluctuations of IDPs are substantially reduced after being stretched. This design may serve as a platform for single-molecule protein analysis with reduced thermal noise.

Visualization of Intrinsically Disordered Regions of Proteins by High-Speed Atomic Force Microscopy

ChemPhysChem ◽  
2008 ◽  
Vol 9 (13) ◽  
pp. 1859-1866 ◽  
Author(s):  
Atsushi Miyagi ◽  
Yasuo Tsunaka ◽  
Takayuki Uchihashi ◽  
Kouta Mayanagi ◽  
Susumu Hirose ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
High Speed ◽  
Force Microscopy ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Atomic Force ◽  
Disordered Regions

scholarly journals Dynamics of oligomer and amyloid fibril formation by yeast prion Sup35 observed by high-speed atomic force microscopy

2020 ◽  
Vol 117 (14) ◽  
pp. 7831-7836 ◽  
Author(s):  
Hiroki Konno ◽  
Takahiro Watanabe-Nakayama ◽  
Takayuki Uchihashi ◽  
Momoko Okuda ◽  
Liwen Zhu ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
High Speed ◽  
Amyloid Fibrils ◽  
Yeast Prion ◽  
Force Microscopy ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Atomic Force ◽  
Freely Moving ◽  
Disordered Regions

The yeast prion protein Sup35, which contains intrinsically disordered regions, forms amyloid fibrils responsible for a prion phenotype [PSI+]. Using high-speed atomic force microscopy (HS-AFM), we directly visualized the prion determinant domain (Sup35NM) and the formation of its oligomers and fibrils at subsecond and submolecular resolutions. Monomers with freely moving tail-like regions initially appeared in the images, and subsequently oligomers with distinct sizes of ∼1.7 and 3 to 4 nm progressively accumulated. Nevertheless, these oligomers did not form fibrils, even after an incubation for 2 h in the presence of monomers. Fibrils appeared after much longer monomer incubation. The fibril elongation occurred smoothly without discrete steps, suggesting gradual conversions of the incorporated monomers into cross-β structures. The individual oligomers were separated from each other and also from the fibrils by respective, identical lengths on the mica surface, probably due to repulsion caused by the freely moving disordered regions. Based on these HS-AFM observations, we propose that the freely moving tails of the monomers are incorporated into the fibril ends, and then the structural conversions to cross-β structures gradually occur.

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