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 Screening for Influx through Membrane Proteins on the Single-Molecule Level using an Automated and Parallel Lipid Bilayer Platform

2014 ◽  
Vol 106 (2) ◽  
pp. 559a
Author(s):  
Mohamed Kreir ◽  
Matthias Beckler ◽  
Astrid Seifert ◽  
Conrad Weichbrodt ◽  
Gerhard Baaken ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Lipid Bilayer ◽  
Single Molecule ◽  
Single Molecule Level

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 Directed manipulation of membrane proteins by fluorescent magnetic nanoparticles

2020 ◽  
Author(s):  
Jia Hui Li ◽  
Paula Santos-Otte ◽  
Braedyn Au ◽  
Jakob Rentsch ◽  
Stephan Block ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Magnetic Nanoparticles ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Single Molecule ◽  
Super Resolution ◽  
Single Particle Tracking ◽  
Live Cells ◽  
Single Molecule Level ◽  
Membrane Components

AbstractThe plasma membrane is the interface through which cells interact with their environment. Membrane proteins are embedded in the lipid bilayer of the plasma membrane and their function in this context is often linked to their specific location and dynamics within the membrane. However, few methods are available for nanoscale manipulation of membrane protein location at the single molecule level. Here, we report the use of fluorescent magnetic nanoparticles (FMNPs) to track membrane molecules and to manipulate their movement. FMNPs allow single-particle tracking (SPT) at 10 nm spatial and 5 ms temporal resolution, and using a magnetic needle, we pull membrane components laterally through the membrane with femtonewton-range forces. In this way, we successfully dragged lipid-anchored and transmembrane proteins over the surface of living cells. Doing so, we detected submembrane barriers and in combination with super-resolution microscopy could localize these barriers to the actin cytoskeleton. We present here a versatile approach to probe membrane processes in live cells via the magnetic control of membrane protein motion.

scholarly journals Semisynthetic protein nanoreactor for single-molecule chemistry

2015 ◽  
Vol 112 (45) ◽  
pp. 13768-13773 ◽  
Author(s):  
Joongoo Lee ◽  
Hagan Bayley
Keyword(s):  
Amino Acid ◽  
Single Molecule ◽  
Solid Phase ◽  
Solid Phase Peptide Synthesis ◽  
Ionic Current ◽  
Unnatural Amino Acid ◽  
Chemical Ligation ◽  
Single Molecule Level ◽  
Flow Through ◽  
Recombinant Polypeptides

The covalent chemistry of individual reactants bound within a protein pore can be monitored by observing the ionic current flow through the pore, which acts as a nanoreactor responding to bond-making and bond-breaking events. In the present work, we incorporated an unnatural amino acid into the α-hemolysin (αHL) pore by using solid-phase peptide synthesis to make the central segment of the polypeptide chain, which forms the transmembrane β-barrel of the assembled heptamer. The full-length αHL monomer was obtained by native chemical ligation of the central synthetic peptide to flanking recombinant polypeptides. αHL pores with one semisynthetic subunit were then used as nanoreactors for single-molecule chemistry. By introducing an amino acid with a terminal alkyne group, we were able to visualize click chemistry at the single-molecule level, which revealed a long-lived (4.5-s) reaction intermediate. Additional side chains might be introduced in a similar fashion, thereby greatly expanding the range of single-molecule covalent chemistry that can be investigated by the nanoreactor approach.

scholarly journals Conformation switching of single native proteins revealed by nanomechanical probing without a pulling force

Nanoscale ◽  
2019 ◽  
Vol 11 (42) ◽  
pp. 19933-19942
Author(s):  
Fabiola A. Gutiérrez-Mejía ◽  
Christian P. Moerland ◽  
Leo J. van IJzendoorn ◽  
Menno W. J. Prins
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Biological Function ◽  
Single Molecule Level ◽  
Native Proteins ◽  
Protein Conformational Changes ◽  
P Protein ◽  
Heterogeneous Nature ◽  
Pulling Force

Protein conformational changes are essential to biological function, and the heterogeneous nature of the corresponding protein states provokes an interest to measure conformational changes at the single molecule level.

In situ fluorescent profiling of living cell membrane proteins at a single-molecule level

2019 ◽  
Vol 55 (28) ◽  
pp. 4043-4046 ◽  
Author(s):  
Yuanyuan Fan ◽  
Lu Li ◽  
Meng Lu ◽  
Haibin Si ◽  
Bo Tang
Keyword(s):  
Membrane Proteins ◽  
Cell Membrane ◽  
Single Molecule ◽  
Signal Amplification ◽  
Living Cells ◽  
Single Molecule Level ◽  
Visualization Analysis ◽  
Amplification Method ◽  
Cell Membrane Proteins

A signal amplification method is developed for visualization analysis of membrane proteins on living cells at a single-molecule level.

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