scholarly journals High-speed AFM reveals accelerated binding of agitoxin-2 to a K+ channel by induced fit

2019 ◽  
Vol 5 (7) ◽  
pp. eaax0495 ◽  
Author(s):  
A. Sumino ◽  
T. Sumikama ◽  
T. Uchihashi ◽  
S. Oiki
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Scorpion Venom ◽  
State Model ◽  
K Channel ◽  
Induced Fit ◽  
Force Microscopy ◽  
Toxin Binding ◽  
Docking Model ◽  
Antigen Antibody

Agitoxin-2 (AgTx2) from scorpion venom is a potent blocker of K+ channels. The docking model has been elucidated, but it remains unclear whether binding dynamics are described by a two-state model (AgTx2-bound and AgTx2-unbound) or a more complicated mechanism, such as induced fit or conformational selection. Here, we observed the binding dynamics of AgTx2 to the KcsA channel using high-speed atomic force microscopy. From images of repeated binding and dissociation of AgTx2 to the channel, single-molecule kinetic analyses revealed that the affinity of the channel for AgTx2 increased during persistent binding and decreased during persistent dissociation. We propose a four-state model, including high- and low-affinity states of the channel, with relevant rate constants. An induced-fit pathway was dominant and accelerated binding by 400 times. This is the first analytical imaging of scorpion toxin binding in real time, which is applicable to various biological dynamics including channel ligands, DNA-modifier proteins, and antigen-antibody complexes.

scholarly journals Steric blockage of lysenin toxin by crowding

2020 ◽  
Author(s):  
Ignacio L.B. Munguira ◽  
Alfonso Barbas
Keyword(s):  
Lipid Bilayers ◽  
Single Molecule ◽  
Pore Formation ◽  
High Speed ◽  
Protein Activity ◽  
Force Microscopy ◽  
Toxin Binding ◽  
Binding Domains ◽  
Ph Decrease ◽  
Immune Defences

AbstractIncreasing evidence signals the importance of macromolecular crowding on the regulation of the membrane protein activity. Lysenin is a pore forming toxin that forms crowded assemblies in membrane containing sphingomyelin microdomains. We studied the role of crowding on the activity of Lysenin thanks to High Speed Atomic Force Microscopy. In this study we show that pore formation requires available space around to take place, being sterically block in crowded environments, and verified it with non-Supported Lipid Bilayers mimicking the mechanical conditions of cell membranes. A continuous pH decrease and a single molecule compression experiments show that pore formation liberates membrane space leading to prepore-to-pore transitions. The study of the effects of prepore insertion comparing pore formation induced by sudden pH decrease in lysenin assemblies with thousand simulations show that liberation of space unblocks pore formation and could contribute to elude the cellular non-immune defences. Based on our findings we propose a refinement of current prepore structure and insertion models. We envision novel antibiotic strategies based on toxin-binding-domains “crowders”.

scholarly journals A molecularly engineered, broad-spectrum anti-coronavirus lectin inhibits SARS-CoV-2 and MERS-CoV infection in vivo

2021 ◽  
Author(s):  
David Markovitz ◽  
Jasper Chan ◽  
Yoo Jin Oh ◽  
Shuofeng Yuan ◽  
Hin Chu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Broad Spectrum ◽  
High Speed ◽  
Viral Infections ◽  
Healthy Human ◽  
Force Microscopy ◽  
Pulmonary Damage ◽  
High Mannose ◽  
Viral Components

Abstract Coronaviruses have repeatedly crossed species barriers to cause epidemics1. “Pan-coronavirus” antivirals targeting conserved viral components involved in coronavirus replication, such as the extensively glycosylated spike protein, can be designed. Here we show that the rationally engineered H84T-banana lectin (H84T-BanLec), which specifically recognizes high-mannose found on viral proteins but seldom on healthy human cells2, potently inhibits the highly virulent MERS-CoV, pandemic SARS-CoV-2 and its variants, and other human-pathogenic coronaviruses at nanomolar concentrations. MERS-CoV-infected human DPP4-transgenic mice treated by H84T-BanLec have significantly higher survival, lower viral burden, and reduced pulmonary damage. Similarly, prophylactic or therapeutic H84T-BanLec is effective against SARS-CoV-2 in hamsters. Importantly, intranasally and intraperitoneally administered H84T-BanLec are comparably effective. Time-of-drug-addition assay shows that H84T-BanLec targets virus entry. Real-time structural analysis with high-speed atomic force microscopy depicts multi-molecular associations of H84T-BanLec dimers with the SARS-CoV-2 spike trimer. Single-molecule force spectroscopy demonstrates binding of H84T-BanLec to multiple SARS-CoV-2 spike mannose sites with high affinity, and that H84T-BanLec competes with SARS-CoV-2 spike for binding to cellular ACE2. Modelling experiments identify distinct high-mannose glycans in spike recognized by H84T-BanLec. The multiple H84T-BanLec binding sites on spike likely account for the activity against SARS-CoV-2 variants and the lack of resistant mutants. The broad-spectrum H84T-BanLec should be clinically evaluated in respiratory viral infections including COVID-19.

scholarly journals Resolving the data asynchronicity in high-speed atomic force microscopy measurement via the Kalman Smoother

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Shintaroh Kubo ◽  
Suguru Kato ◽  
Kazuyuki Nakamura ◽  
Noriyuki Kodera ◽  
Shoji Takada
Keyword(s):  
Atomic Force Microscopy ◽  
Kalman Filter ◽  
Single Molecule ◽  
High Speed ◽  
Ground Truth ◽  
Bayesian Filtering ◽  
Single Molecule Level ◽  
Force Microscopy ◽  
Kalman Smoother ◽  
Atomic Force

Abstract High-speed atomic force microscopy (HS-AFM) is a scanning probe microscopy that can capture structural dynamics of biomolecules in real time at single molecule level near physiological condition. Albeit much improvement, while scanning one frame of HS-AFM movies, biomolecules often change their conformations largely. Thus, the obtained frame images can be hampered by the time-difference, the asynchronicity, in the data acquisition. Here, to resolve this data asynchronicity in the HS-AFM movie, we developed Kalman filter and smoother methods, some of the sequential Bayesian filtering approaches. The Kalman filter/smoother methods use alternative steps of a short time-propagation by a linear dynamical system and a correction by the likelihood of AFM data acquired pixel by pixel. We first tested the method using a toy model of a diffusing cone, showing that the Kalman smoother method outperforms to reproduce the ground-truth movie. We then applied the Kalman smoother to a synthetic movie for conformational change dynamics of a motor protein, i.e., dynein, confirming the superiority of the Kalman smoother. Finally, we applied the Kalman smoother to two real HS-AFM movies, FlhAC and centralspindlin, reducing distortion and noise in the AFM movies. The method is general and can be applied to any HS-AFM movies.

scholarly journals Single-Molecule Dynamics of the DNA−EcoRII Protein Complexes Revealed with High-Speed Atomic Force Microscopy

Biochemistry ◽  
2009 ◽  
Vol 48 (44) ◽  
pp. 10492-10498 ◽  
Author(s):  
Jamie L. Gilmore ◽  
Yuki Suzuki ◽  
Gintautas Tamulaitis ◽  
Virginijus Siksnys ◽  
Kunio Takeyasu ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Single Molecule ◽  
High Speed ◽  
Protein Complexes ◽  
Force Microscopy ◽  
Atomic Force ◽  
Single Molecule Dynamics ◽  
Molecule Dynamics

scholarly journals Mobility of nucleosomes is regulated by their binding partners

2022 ◽  
Author(s):  
Daniel P Melters ◽  
Keir C Neuman ◽  
Tatini Rakshit ◽  
Yamini Dalal
Keyword(s):  
Atomic Force Microscopy ◽  
Single Molecule ◽  
High Speed ◽  
Diffusion Constant ◽  
Chromatin Accessibility ◽  
Chromatin Dynamics ◽  
Single Molecule Level ◽  
Force Microscopy ◽  
Atomic Force

Chromatin accessibility is modulated in a variety of ways, both to create open and closed chromatin states which are critical for eukaryotic gene regulation. At the mechanistic single molecule level, how accessibility is regulated remains a fundamental question in the field. Here, we use single molecule tracking by high-speed atomic force microscopy to investigate this question using chromatin arrays and extend our findings into the nucleus. By high-speed atomic force microscopy, we tracked chromatin dynamics in real time and observed that the essential kinetochore protein CENP-C reduces the diffusion constant of CENP-A nucleosomes and the linker H1.5 protein restricts H3 nucleosome mobility. We subsequently interrogated how CENP-C modulates CENP-A chromatin dynamics in vivo. Overexpressing CENP-C resulted in reduced centromeric transcription and impaired loading of new CENP-A molecules. These data suggest a model in which inner kinetochore proteins are critically involved in modulating chromatin accessibility and consequently, noncoding transcription at human centromeres.

scholarly journals Steric blockage of lysenin toxin by crowding

2020 ◽  
Author(s):  
Ignacio L.B. Munguira ◽  
Alfonso Barbas
Keyword(s):  
Lipid Bilayers ◽  
Single Molecule ◽  
Pore Formation ◽  
High Speed ◽  
Protein Activity ◽  
Force Microscopy ◽  
Atomic Force ◽  
Ph Decrease ◽  
Immune Defences ◽  
Crowded Environments

Abstract Increasing evidence signals the importance of macromolecular crowding on the regulation of the membrane protein activity. Lysenin is a pore forming toxin that forms crowded assemblies in membrane containing sphingomyelin microdomains. We studied the role of crowding on the activity of Lysenin thanks to High Speed Atomic Force Microscopy. In this study we show that pore formation requires available space around to take place, being sterically block in crowded environments, and verified it with nonSupported Lipid Bilayers mimicking the mechanical conditions of cell membranes. A continuous pH decrease and a single molecule compression experiments show that pore formation liberates membrane space leading to prepore-to-pore transitions. The study of the effects of prepore insertion comparing pore formation induced by sudden pH decrease in lysenin assemblies with thousand simulations show that liberation of space unblocks pore formation and could contribute to elude the cellular non-immune defences. Based on our findings we propose a refinement of current prepore structure and insertion models. We envision novel antibiotic strategies based on toxin-bindingdomains crowders.

Quantum-dot antibody conjugation visualized at the single-molecule scale with high-speed atomic force microscopy

2018 ◽  
Vol 167 ◽  
pp. 267-274 ◽  
Author(s):  
Takayuki Umakoshi ◽  
Hikari Udaka ◽  
Takayuki Uchihashi ◽  
Toshio Ando ◽  
Miho Suzuki ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Quantum Dot ◽  
Single Molecule ◽  
High Speed ◽  
Force Microscopy ◽  
Atomic Force ◽  
Antibody Conjugation
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