scholarly journals AFM-Based Force Spectroscopy Guided by Recognition Imaging: A New Mode for Mapping and Studying Interaction Sites at Low Lateral Density

2019 ◽  
Vol 2 (1) ◽  
pp. 6 ◽  
Author(s):  
Melanie Koehler ◽  
Anny Fis ◽  
Hermann J. Gruber ◽  
Peter Hinterdorfer
Keyword(s):  
Single Molecule ◽  
Lipid Membrane ◽  
Force Spectroscopy ◽  
Regulatory Elements ◽  
Membrane Receptors ◽  
Ligand Molecule ◽  
Dynamic Force ◽  
Receptor Ligand ◽  
Interaction Sites ◽  
Recognition Imaging

Ligand binding to receptors is one of the most important regulatory elements in biology as it is the initiating step in signaling pathways and cascades. Thus, precisely localizing binding sites and measuring interaction forces between cognate receptor–ligand pairs leads to new insights into the molecular recognition involved in these processes. Here we present a detailed protocol about applying a technique, which combines atomic force microscopy (AFM)-based recognition imaging and force spectroscopy for studying the interaction between (membrane) receptors and ligands on the single molecule level. This method allows for the selection of a single receptor molecule reconstituted into a supported lipid membrane at low density, with the subsequent quantification of the receptor–ligand unbinding force. Based on AFM tapping mode, a cantilever tip carrying a ligand molecule is oscillated across a membrane. Topography and recognition images of reconstituted receptors are recorded simultaneously by analyzing the downward and upward parts of the oscillation, respectively. Functional receptor molecules are selected from the recognition image with nanometer resolution before the AFM is switched to the force spectroscopy mode, using positional feedback control. The combined mode allows for dynamic force probing on different pre-selected molecules. This strategy results in higher throughput when compared with force mapping. Applied to two different receptor–ligand pairs, we validated the presented new mode.

Interpretations of Receptor–Ligand Dissociation Kinetics from Single-Molecule Pulling Experiments

2017 ◽  
Vol 09 (03) ◽  
pp. 1750042 ◽  
Author(s):  
Yuan Feng ◽  
Ji Lin ◽  
Jin Qian
Keyword(s):  
Single Molecule ◽  
Gaussian Approximation ◽  
Force Spectroscopy ◽  
Thermal Fluctuations ◽  
Theoretical Issue ◽  
Dynamic Force ◽  
Dynamic Force Spectroscopy ◽  
Receptor Ligand ◽  
Bond Dissociation ◽  
Force Relation

The kinetic response of receptor–ligand bonds to externally imposed force is of essential importance for adhesion-mediated behaviors of cells. Two prevailing experimental approaches, so-called dynamic force spectroscopy and force clamp assay, have been commonly adopted to probe the force dependence of bond dissociation rate at single-molecule level. This study focuses on the outstanding theoretical issue concerning the distinct loading paths and different procedures to extract the kinetic information in the two types of experiments. To address the issue, Monte Carlo simulations have been performed to simulate bond dissociation as a well-to-barrier escape process under dynamically imposed force as well as thermal fluctuations, and the consistency of quantitative interpretations on force-dependent bond lifetimes from the different approaches is examined. Our numerical results show that all the interpretations from different methods collapse into a single master curve of lifetime–force relation for receptor–ligand bonds with slip behavior. However, for bonds with biphasic catch–slip behavior, a procedure based on a Gaussian approximation of rupture force distributions, proposed by Dudko for dynamic force spectroscopy, tends to underestimate bond lifetime for certain force range.

scholarly journals Dissecting the cytochrome c2–reaction centre interaction in bacterial photosynthesis using single molecule force spectroscopy

2019 ◽  
Vol 476 (15) ◽  
pp. 2173-2190
Author(s):  
Cvetelin Vasilev ◽  
Guy E. Mayneord ◽  
Amanda A. Brindley ◽  
Matthew P. Johnson ◽  
C. Neil Hunter
Keyword(s):  
Single Molecule ◽  
Silicon Oxide ◽  
Fluorescent Protein ◽  
Dissociation Constants ◽  
Force Spectroscopy ◽  
Spectroscopic Studies ◽  
Bacterial Photosynthesis ◽  
Dynamic Force ◽  
Reaction Centre ◽  
Cytochrome C2

Abstract The reversible docking of small, diffusible redox proteins onto a membrane protein complex is a common feature of bacterial, mitochondrial and photosynthetic electron transfer (ET) chains. Spectroscopic studies of ensembles of such redox partners have been used to determine ET rates and dissociation constants. Here, we report a single-molecule analysis of the forces that stabilise transient ET complexes. We examined the interaction of two components of bacterial photosynthesis, cytochrome c2 and the reaction centre (RC) complex, using dynamic force spectroscopy and PeakForce quantitative nanomechanical imaging. RC–LH1–PufX complexes, attached to silicon nitride AFM probes and maintained in a photo-oxidised state, were lowered onto a silicon oxide substrate bearing dispersed, immobilised and reduced cytochrome c2 molecules. Microscale patterns of cytochrome c2 and the cyan fluorescent protein were used to validate the specificity of recognition between tip-attached RCs and surface-tethered cytochrome c2. Following the transient association of photo-oxidised RC and reduced cytochrome c2 molecules, retraction of the RC-functionalised probe met with resistance, and forces between 112 and 887 pN were required to disrupt the post-ET RC–c2 complex, depending on the retraction velocities used. If tip-attached RCs were reduced instead, the probability of interaction with reduced cytochrome c2 molecules decreased 5-fold. Thus, the redox states of the cytochrome c2 haem cofactor and RC ‘special pair’ bacteriochlorophyll dimer are important for establishing a productive ET complex. The millisecond persistence of the post-ET cytochrome c2[oxidised]–RC[reduced] ‘product’ state is compatible with rates of cyclic photosynthetic ET, at physiologically relevant light intensities.

Dynamic force spectroscopy for quantifying single-molecule organo–mineral interactions

CrystEngComm ◽  
2021 ◽  
Vol 23 (1) ◽  
pp. 11-23
Author(s):  
Hang Zhai ◽  
Wenjun Zhang ◽  
Lijun Wang ◽  
Christine V. Putnis
Keyword(s):  
Single Molecule ◽  
Force Spectroscopy ◽  
Nucleation And Growth ◽  
Crystal Nucleation ◽  
Dynamic Force ◽  
Dynamic Force Spectroscopy ◽  
Crystal Phases

Organo–mineral interactions have long been the focus in the fields of biomineralization and geomineralization, since such interactions not only modulate the dynamics of crystal nucleation and growth but may also change crystal phases, morphologies, and structures.

Energy landscapes of receptor–ligand bonds explored with dynamic force spectroscopy

Nature ◽  
10.1038/16219 ◽  
1999 ◽  
Vol 397 (6714) ◽  
pp. 50-53 ◽  
Author(s):  
R. Merkel ◽  
P. Nassoy ◽  
A. Leung ◽  
K. Ritchie ◽  
E. Evans
Keyword(s):  
Force Spectroscopy ◽  
Dynamic Force ◽  
Energy Landscapes ◽  
Dynamic Force Spectroscopy ◽  
Receptor Ligand

Single-molecule force spectroscopy on polyproteins and receptor–ligand complexes: The current toolbox

2017 ◽  
Vol 197 (1) ◽  
pp. 3-12 ◽  
Author(s):  
Wolfgang Ott ◽  
Markus A. Jobst ◽  
Constantin Schoeler ◽  
Hermann E. Gaub ◽  
Michael A. Nash
Keyword(s):  
Single Molecule ◽  
Force Spectroscopy ◽  
Single Molecule Force Spectroscopy ◽  
Receptor Ligand

Transverse Dynamic Force Spectroscopy:  A Novel Approach to Determining the Complex Stiffness of a Single Molecule

Langmuir ◽  
2002 ◽  
Vol 18 (5) ◽  
pp. 1729-1733 ◽  
Author(s):  
A. D. L. Humphris ◽  
M. Antognozzi ◽  
T. J. McMaster ◽  
M. J. Miles
Keyword(s):  
Single Molecule ◽  
Force Spectroscopy ◽  
Dynamic Force ◽  
Dynamic Force Spectroscopy ◽  
Transverse Dynamic ◽  
Novel Approach
Sign in / Sign up

Export Citation Format

Share Document