scholarly journals Single-molecule nanopore sensing of actin dynamics and drug binding

2019 ◽  
Author(s):  
Xiaoyi Wang ◽  
Mark D. Wilkinson ◽  
Xiaoyan Lin ◽  
Ren Ren ◽  
Keith Willison ◽  
...  
Keyword(s):  
Protein Folding ◽  
Single Molecule ◽  
Eukaryotic Cell ◽  
Molecular State ◽  
Drug Binding ◽  
Actin Dynamics ◽  
Label Free ◽  
Single Molecule Level ◽  
Native Proteins ◽  
Voltage Dependent

AbstractActin is a key protein in the dynamic processes within the eukaryotic cell. To date, methods exploring the molecular state of actin are limited to insights gained from structural approaches, providing a snapshot of protein folding, or methods that require chemical modifications compromising actin monomer thermostability. Nanopore sensing permits label-free investigation of native proteins and is ideally suited to study proteins such as actin that require specialised buffers and cofactors. Using nanopores we determined the state of actin at the macromolecular level (filamentous or globular) and in its monomeric form bound to inhibitors. We revealed urea-dependent and voltage-dependent transitional states and observed unfolding process within which sub-populations of transient actin oligomers are visible. We detected, in real-time, drug-binding and filament-growth events at the single-molecule level. This enabled us to calculate binding stoichiometries and to propose a model for protein dynamics using unmodified, native actin molecules, demostrating the promise of nanopores sensing for in-depth understanding of protein folding landscapes and for drug discovery.

scholarly journals Single-molecule nanopore enzymology

2017 ◽  
Vol 372 (1726) ◽  
pp. 20160230 ◽  
Author(s):  
Kherim Willems ◽  
Veerle Van Meervelt ◽  
Carsten Wloka ◽  
Giovanni Maglia
Keyword(s):  
Membrane Proteins ◽  
Chemical Reactions ◽  
Bias Voltage ◽  
Single Molecule ◽  
Ionic Current ◽  
Enzymatic Reactions ◽  
Current Passing ◽  
Single Molecule Level ◽  
Membrane Pores ◽  
Native Proteins

Biological nanopores are a class of membrane proteins that open nanoscale water conduits in biological membranes. When they are reconstituted in artificial membranes and a bias voltage is applied across the membrane, the ionic current passing through individual nanopores can be used to monitor chemical reactions, to recognize individual molecules and, of most interest, to sequence DNA. In addition, a more recent nanopore application is the analysis of single proteins and enzymes. Monitoring enzymatic reactions with nanopores, i.e. nanopore enzymology, has the unique advantage that it allows long-timescale observations of native proteins at the single-molecule level. Here, we describe the approaches and challenges in nanopore enzymology. This article is part of the themed issue ‘Membrane pores: from structure and assembly, to medicine and technology’.

scholarly journals Label-Free Visualisation of Actin Nucleation and Polymerisation at the Single-Molecule Level using Interferometric Scattering Microscopy

2018 ◽  
Vol 114 (3) ◽  
pp. 381a
Author(s):  
Nikolas Hundt ◽  
Andrew Tyler ◽  
Gavin Young ◽  
Daniel Cole ◽  
Adam J. Fineberg ◽  
...  
Keyword(s):  
Single Molecule ◽  
Label Free ◽  
Actin Nucleation ◽  
Single Molecule Level

scholarly journals Chaperone-Mediated Mechanical Protein Folding at the Single Molecule Level

2016 ◽  
Vol 110 (3) ◽  
pp. 392a
Author(s):  
Judit Perales-Calvo ◽  
David Giganti ◽  
Sergi Garcia-Manyes
Keyword(s):  
Protein Folding ◽  
Single Molecule ◽  
Single Molecule Level

scholarly journals Quantifying protein-protein interactions by molecular counting with mass photometry

2020 ◽  
Author(s):  
Fabian Soltermann ◽  
Eric D.B. Foley ◽  
Veronica Pagnoni ◽  
Martin R. Galpin ◽  
Justin L.P. Benesch ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Single Molecule ◽  
Protein Interactions ◽  
Analytical Techniques ◽  
Binding Affinities ◽  
Label Free ◽  
Protein Protein Interactions ◽  
Single Molecule Level ◽  
Minimal Invasiveness ◽  
Universal Approach

AbstractInteractions between biomolecules control the processes of life in health, and their malfunction in disease, making their characterization and quantification essential. Immobilization- and label-free analytical techniques are particular desirable because of their simplicity and minimal invasiveness, but struggle to quantify tight interactions. Here, we show that we can accurately count, distinguish by molecular mass, and thereby reveal the relative abundances of different un-labelled biomolecules and their complexes in mixtures at the single-molecule level by mass photometry. These measurements enable us to quantify binding affinities over four orders of magnitude at equilibrium for both simple and complex stoichiometries within minutes, as well as to determine the associated kinetics. Our results introduce mass photometry as a rapid, simple and label-free method for studying sub-μM binding affinities, with potential to be extended towards a universal approach for characterising complex biomolecular interactions.

scholarly journals Revisiting Protein Folding at the Single Molecule Level

2009 ◽  
Vol 96 (3) ◽  
pp. 217a
Author(s):  
Sergi Garcia-Manyes ◽  
Lorna Dougan ◽  
Carmen L. Badilla ◽  
Jasna Brujic ◽  
Julio Fernandez
Keyword(s):  
Protein Folding ◽  
Single Molecule ◽  
Single Molecule Level

scholarly journals A functional family of fluorescent nucleotide analogues to investigate actin dynamics and energetics

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jessica Colombo ◽  
Adrien Antkowiak ◽  
Konstantin Kogan ◽  
Tommi Kotila ◽  
Jenna Elliott ◽  
...  
Keyword(s):  
Single Molecule ◽  
Actin Polymerization ◽  
Eukaryotic Cell ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Actin Assembly ◽  
Nucleotide Exchange ◽  
Nucleotide Analogues ◽  
Highly Sensitive ◽  
Fluorescent Nucleotides

AbstractActin polymerization provides force for vital processes of the eukaryotic cell, but our understanding of actin dynamics and energetics remains limited due to the lack of high-quality probes. Most current probes affect dynamics of actin or its interactions with actin-binding proteins (ABPs), and cannot track the bound nucleotide. Here, we identify a family of highly sensitive fluorescent nucleotide analogues structurally compatible with actin. We demonstrate that these fluorescent nucleotides bind to actin, maintain functional interactions with a number of essential ABPs, are hydrolyzed within actin filaments, and provide energy to power actin-based processes. These probes also enable monitoring actin assembly and nucleotide exchange with single-molecule microscopy and fluorescence anisotropy kinetics, therefore providing robust and highly versatile tools to study actin dynamics and functions of ABPs.

scholarly journals Cellular Lensing and Near Infrared Fluorescent Nanosensor Arrays to Enable Chemical Efflux Cytometry

2020 ◽  
Author(s):  
Soo-Yeon Cho ◽  
Xun Gong ◽  
Volodymyr Koman ◽  
Matthias Kuehne ◽  
Sun Jin Moon ◽  
...  
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Near Infrared ◽  
Microfluidic Channel ◽  
Human Monocyte ◽  
Cellular Heterogeneity ◽  
Label Free ◽  
Temporal Precision ◽  
Single Molecule Level ◽  
Chemical Cytometry

Abstract Nanosensor have proven to be powerful tools to monitor single biological cells and organisms, achieving spatial and temporal precision even at the single molecule level. However, there has not been a way of extending this approach to statistically relevant numbers of living cells and organisms. Herein, we design and fabricate a high throughput nanosensor array in a microfluidic channel that addresses this limitation, creating a Nanosensor Chemical Cytometry (NCC). An array of nIR fluorescent single walled carbon nanotube (SWNT) nanosensors is integrated along a microfluidic channel through which a population of flowing cells is guided. We show that one can utilize the flowing cell itself as highly informative Gaussian lenses projecting nIR emission profiles and extract rich information on a per cell basis at high throughput. This unique biophotonic waveguide allows for quantified cross-correlation of the biomolecular information with physical properties such as cellular diameter, refractive index (RI), and eccentricity and creates a label-free chemical cytometer for the measurement of cellular heterogeneity with unprecedented precision. As an example, the NCC can profile the immune response heterogeneities of distinct human monocyte populations at attomolar (10-18 moles) sensitivity in a completely non-destructive and real-time manner with a rate of ~100 cells/frame, highest range demonstrated to date for state of the art chemical cytometry. We demonstrate distinct H2O2 efflux heterogeneities between 330 and 624 attomole/cell·min with cell projected areas between 271 and 263 µm2, eccentricity values between 0.405 and 0.363 and RI values between 1.383 and 1.377 for non-activated and activated human monocytes, respectively. Hence, we show that our nanotechnology based biophotonic cytometer has significant potential and versatility to answer important questions and provide new insight in immunology, cell manufacturing and biopharmaceutical research.

scholarly journals A label-free approach to detect ligand binding to cell surface proteins in real time

2018 ◽  
Author(s):  
Verena Burtscher ◽  
Matej Hotka ◽  
Yang Li ◽  
Michael Freissmuth ◽  
Walter Sandtner
Keyword(s):  
Ligand Binding ◽  
Real Time ◽  
Single Molecule ◽  
Membrane Capacitance ◽  
Surface Proteins ◽  
Displacement Current ◽  
High Temporal Resolution ◽  
Label Free ◽  
Single Molecule Level ◽  
Electrophysiological Recordings

AbstractElectrophysiological recordings allow for monitoring the operation of proteins with high temporal resolution down to the single molecule level. This technique has been exploited to track either ion flow arising from channel opening or the synchronized movement of charged residues and/or ions within the membrane electric field. Here, we describe a novel type of current by using the serotonin transporter (SERT) as a model. We examined transient currents elicited on rapid application of specific SERT inhibitors. Our analysis shows that these currents originate from ligand binding and not from a conformational change. The Gouy-Chapman model predicts that a ligand-induced elimination/neutralization of surface charge must produce a displacement current and related apparent changes in membrane capacitance. Here we verified these predictions with SERT. Our observations demonstrate that ligand binding to a protein can be monitored in real time and in a label-free manner by recording the membrane capacitance.

scholarly journals PCR-free, label-free detection of sequence-specific DNA with single-molecule sensitivity using in vitro N-hybrid system in microfluidic drops

2022 ◽  
Author(s):  
Yizhe Zhang ◽  
David A Weitz
Keyword(s):  
Single Molecule ◽  
High Specificity ◽  
Label Free ◽  
Hybrid Strategy ◽  
Single Molecule Level ◽  
Target Dna ◽  
Label Free Detection ◽  
Novel Method ◽  
Picoliter Droplet

We propose a novel method that can detect DNA with high specificity at the single-molecule level by employing the in vitro N-hybrid strategy realized in sub-picoliter microfluidic drops. It detects target DNA based on the specific interactions of the target-encoded proteins with their partner molecules, and achieves single-molecule sensitivity via signal-transduction and signal-amplification during gene-expression processes in a sub-picoliter droplet, therefore effectively avoiding complicated procedures in labeling-based methods or biases and artifacts in PCR-based methods.

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