scholarly journals Single molecule kinetics of bacteriorhodopsin by HS-AFM

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Alma P. Perrino ◽  
Atsushi Miyagi ◽  
Simon Scheuring
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
High Speed ◽  
Continuous Light ◽  
Photon Absorption ◽  
Short Pulses ◽  
Reaction Cycle ◽  
Force Microscopy ◽  
Molecule Reaction ◽  
Kinetics Of

AbstractBacteriorhodopsin is a seven-helix light-driven proton-pump that was structurally and functionally extensively studied. Despite a wealth of data, the single molecule kinetics of the reaction cycle remain unknown. Here, we use high-speed atomic force microscopy methods to characterize the single molecule kinetics of wild-type bR exposed to continuous light and short pulses. Monitoring bR conformational changes with millisecond temporal resolution, we determine that the cytoplasmic gate opens 2.9 ms after photon absorption, and stays open for proton capture for 13.2 ms. Surprisingly, a previously active protomer cannot be reactivated for another 37.6 ms, even under excess continuous light, giving a single molecule reaction cycle of ~20 s−1. The reaction cycle slows at low light where the closed state is prolonged, and at basic or acidic pH where the open state is extended.

scholarly journals Real-time visualization of conformational changes within single MloK1 cyclic nucleotide-modulated channels

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Martina Rangl ◽  
Atsushi Miyagi ◽  
Julia Kowal ◽  
Henning Stahlberg ◽  
Crina M. Nimigean ◽  
...  
Keyword(s):  
Real Time ◽  
Conformational Changes ◽  
Cyclic Nucleotide ◽  
High Speed ◽  
Function Analysis ◽  
Stable Conformation ◽  
Mesorhizobium Loti ◽  
Force Microscopy ◽  
Atomic Force ◽  
Real Time Visualization

AbstractEukaryotic cyclic nucleotide-modulated (CNM) ion channels perform various physiological roles by opening in response to cyclic nucleotides binding to a specialized cyclic nucleotide-binding domain. Despite progress in structure-function analysis, the conformational rearrangements underlying the gating of these channels are still unknown. Here, we image ligand-induced conformational changes in single CNM channels from Mesorhizobium loti (MloK1) in real-time, using high-speed atomic force microscopy. In the presence of cAMP, most channels are in a stable conformation, but a few molecules dynamically switch back and forth (blink) between at least two conformations with different heights. Upon cAMP depletion, more channels start blinking, with blinking heights increasing over time, suggestive of slow, progressive loss of ligands from the tetramer. We propose that during gating, MloK1 transitions from a set of mobile conformations in the absence to a stable conformation in the presence of ligand and that these conformations are central for gating the pore.

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.

Conformational Changes of the Membrane-Bound ATPase of Bacterial Chromatophores Revealed by Fluorescence Changes of Fluorescamine-Labelled Coupling Factors

1978 ◽  
Vol 33 (5-6) ◽  
pp. 421-427
Author(s):  
Peter Gräber ◽  
Sathamm Saphon
Keyword(s):  
Conformational Changes ◽  
Fluorescence Emission ◽  
Continuous Light ◽  
Coupling Factor ◽  
Single Turnover ◽  
Fluorescence Change ◽  
Transfer Inhibitor ◽  
Membrane Bound ◽  
Coupling Factors ◽  
Kinetics Of

Abstract The solubilized coupling factor (F1) of Rps. sphaeroides chromatophores was allowed to react with fiuorescamine which led to a fluorescence labelled F1 . After reconstitution with the depleted membranes the fluorescence-labelled F1 was shown to restore photophosphorylation in continuous light in a similar way to the non-labelled F1 . In parallel, a decrease of the fluorescence emission of the labelled and reconstituted coupling factor was observed. The solubilized and labelled F1 showed also a fluorescence decrease as the polarity of the medium was increased. In single turnover flashes the fluorescence change was found to be inhibited by an uncoupling agent such as FCCP. The kinetics of the change were sensitive to phosphorylating agents and to an “energy transfer inhibitor” such as venturicidin. It is suggested that the observed fluorescence changes reflect conformational changes of the ATPase enzyme complex.

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 Computational Tool for Ensemble Averaging of Single-Molecule Data

2020 ◽  
Author(s):  
Thomas Blackwell ◽  
W. Tom Stump ◽  
Sarah R. Clippinger ◽  
Michael J. Greenberg
Keyword(s):  
Brownian Motion ◽  
User Interface ◽  
Single Molecule ◽  
Conformational Changes ◽  
Graphical User Interface ◽  
Molecular Motions ◽  
Computational Tool ◽  
Ensemble Averaging ◽  
Mechanochemical Coupling ◽  
Kinetics Of

AbstractMolecular motors couple chemical transitions to conformational changes that perform mechanical work in a wide variety of biological processes. Disruption of this coupling can lead to diseases, and therefore there is a need to accurately measure mechanochemical coupling in motors in both health and disease. Optical tweezers, with nanometer spatial and millisecond temporal resolution, have provided valuable insights into these processes. However, fluctuations due to Brownian motion can make it difficult to precisely resolve these conformational changes. One powerful analysis technique that has improved our ability to accurately measure mechanochemical coupling in motor proteins is ensemble averaging of individual trajectories. Here, we present a user-friendly computational tool, Software for Precise Analysis of Single Molecules (SPASM), for generating ensemble averages of single-molecule data. This tool utilizes several conceptual advances, including optimized procedures for identifying single-molecule interactions and the implementation of a change point algorithm, to more precisely resolve molecular transitions. Using both simulated and experimental data, we demonstrate that these advances allow for accurate determination of the mechanics and kinetics of the myosin working stroke with a smaller set of data. Importantly, we provide our open source MATLAB-based program with a graphical user interface that enables others to readily apply these advances to the analysis of their own data.Statement of SignificanceSingle molecule optical trapping experiments have given unprecedented insights into the mechanisms of molecular machines. Analysis of these experiments is often challenging because Brownian motion-induced fluctuations introduce noise that can obscure molecular motions. A powerful technique for analyzing these noisy traces is ensemble averaging of individual binding interactions, which can uncover information about the mechanics and kinetics of molecular motions that are typically obscured by Brownian motion. Here, we provide an open source, easy-to-use computational tool, SPASM, with a graphical user interface for ensemble averaging of single molecule data. This computational tool utilizes several conceptual advances that significantly improve the accuracy and resolution of ensemble averages, enabling the generation of high-resolution averages from a smaller number of binding interactions.

Nanoscale Analysis of the Effect of Pathogenic Mutations on Polycystin-1

2010 ◽  
Author(s):  
Liang Ma ◽  
Meixiang Xu ◽  
Andres F. Oberhauser
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Structure And Function ◽  
Eukaryotic Cells ◽  
Mechanical Forces ◽  
Force Microscopy ◽  
Physiological Conditions ◽  
Atomic Force ◽  
And Function

The activity of proteins and their complexes often involves the conversion of chemical energy (stored or supplied) into mechanical work through conformational changes. Mechanical forces are also crucial for the regulation of the structure and function of cells and tissues. Thus, the shape of eukaryotic cells is the result of cycles of mechano-sensing, mechano-transduction, and mechano-response. Recently developed single-molecule atomic force microscopy (AFM) techniques can be used to manipulate single molecules, both in real time and under physiological conditions, and are ideally suited to directly quantify the forces involved in both intra- and intermolecular protein interactions. In combination with molecular biology and computer simulations, these techniques have been applied to characterize the unfolding and refolding reactions in a variety of proteins, such as titin (an elastic mechano-sensing protein found in muscle) and polycystin-1 (PC1, a mechanosensor found in the kidney).

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.

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