34 results match your criteria afm force-clamp


AFM force-clamp spectroscopy captures the nanomechanics of the Tad pilus retraction.

Nanoscale Horiz 2021 06;6(6):489-496

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Croix du Sud, 4-5, bte, L7.07.07, Louvain-la-Neuve B-1348, Belgium.

Motorization of bacterial pili is key to generate traction forces to achieve cellular function. The Tad (or Type IVc) pilus from Caulobacter crescentus is a widespread motorized nanomachine crucial for bacterial survival, evolution and virulence. An unusual bifunctional ATPase motor drives Tad pilus retraction, which helps the bacteria to land on target surfaces. Read More

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Single-Molecule Force Spectroscopy on the N2A Element of Titin: Effects of Phosphorylation and CARP.

Front Physiol 2020 18;11:173. Epub 2020 Mar 18.

Department of Cellular & Molecular Medicine, The University of Arizona, Tucson, AZ, United States.

Titin is a large filamentous protein that forms a sarcomeric myofilament with a molecular spring region that develops force in stretched sarcomeres. The molecular spring has a complex make-up that includes the N2A element. This element largely consists of a 104-residue unique sequence (N2A-Us) flanked by immunoglobulin domains (I80 and I81). Read More

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Force clamp approach for characterization of nano-assembly in amyloid beta 42 dimer.

Nanoscale 2019 Jul 18;11(25):12259-12265. Epub 2019 Jun 18.

Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198-6025, USA.

Amyloid β (Aβ) oligomers are formed at the early stages of the amyloidogenesis process and exhibit neurotoxicity. Development of oligomer specific therapeutics requires a detailed understanding of oligomerization processes. Amyloid oligomers exist transiently and single-molecule approaches are capable of characterizing such species. Read More

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Viscoelastic properties of normal and cancerous human breast cells are affected differently by contact to adjacent cells.

Acta Biomater 2017 06 7;55:239-248. Epub 2017 Apr 7.

Institute of Applied Physics, University of Tübingen, Auf der Morgenstelle 10, 72076 Tübingen, Germany. Electronic address:

Malignant transformation drastically alters the mechanical properties of the cell and its response to the surrounding cellular environment. We studied the influence of the physical contact between adjacent cells in an epithelial monolayer on the viscoelastic behavior of normal MCF10A, non-invasive cancerous MCF7, and invasive cancerous MDA-MB-231 human breast cells. Using an atomic force microscopy (AFM) imaging technique termed force clamp force mapping (FCFM) to record images of the viscoelastic material properties, we found that normal MCF10A cells are stiffer and have a lower fluidity at confluent than at sparse density. Read More

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AFM/TIRF force clamp measurements of neurosecretory vesicle tethers reveal characteristic unfolding steps.

PLoS One 2017 21;12(3):e0173993. Epub 2017 Mar 21.

School of Applied and Engineering Physics, Engineering, Cornell University, Ithaca, NY, United States of America.

Although several proteins have been implicated in secretory vesicle tethering, the identity and mechanical properties of the components forming the physical vesicle-plasma membrane link remain unknown. Here we present the first experimental measurements of nanomechanical properties of secretory vesicle-plasma membrane tethers using combined AFM force clamp and TIRF microscopy on membrane sheets from PC12 cells expressing the vesicle marker ANF-eGFP. Application of pulling forces generated tether extensions composed of multiple steps with variable length. Read More

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September 2017

Measuring Force-Induced Dissociation Kinetics of Protein Complexes Using Single-Molecule Atomic Force Microscopy.

Methods Enzymol 2017 11;582:297-320. Epub 2016 Nov 11.

Iowa State University, Ames, IA, United States; Ames Laboratory, U.S. Department of Energy, Ames, IA, United States. Electronic address:

Proteins respond to mechanical force by undergoing conformational changes and altering the kinetics of their interactions. However, the biophysical relationship between mechanical force and the lifetime of protein complexes is not completely understood. In this chapter, we provide a step-by-step tutorial on characterizing the force-dependent regulation of protein interactions using in vitro and in vivo single-molecule force clamp measurements with an atomic force microscope (AFM). Read More

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Non-exponential kinetics of unfolding under a constant force.

J Chem Phys 2016 Nov;145(18):185102

Cavendish Laboratory, University of Cambridge, J.J. Thomson Avenue, Cambridge CB3 0HE, United Kingdom.

We examine the population dynamics of naturally folded globular polymers, with a super-hydrophobic "core" inserted at a prescribed point in the polymer chain, unfolding under an application of external force, as in AFM force-clamp spectroscopy. This acts as a crude model for a large class of folded biomolecules with hydrophobic or hydrogen-bonded cores. We find that the introduction of super-hydrophobic units leads to a stochastic variation in the unfolding rate, even when the positions of the added monomers are fixed. Read More

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November 2016

Dually actuated atomic force microscope with miniaturized magnetic bead-actuators for single-molecule force measurements.

Nanoscale Horiz 2016 Nov 14;1(6):488-495. Epub 2016 Sep 14.

Department of Electrical and Electronics Engineering, Bogazici University, Bebek, 34342 Istanbul, Turkey.

We report a novel atomic force microscopy (AFM) technique with dual actuation capabilities using both piezo and magnetic bead actuation for advanced single-molecule force spectroscopy experiments. The experiments are performed by manipulating magnetic microbeads using an electromagnet against a stationary cantilever. Magnetic actuation has been demonstrated before to actuate cantilevers, but here we keep the cantilever stationary and accomplish actuation via free-manipulated microstructures. Read More

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November 2016

SNARE-mediated membrane fusion trajectories derived from force-clamp experiments.

Proc Natl Acad Sci U S A 2016 11 2;113(46):13051-13056. Epub 2016 Nov 2.

Institute of Physical Chemistry, University of Göttingen, 37077 Goettingen, Germany;

Fusion of lipid bilayers is usually prevented by large energy barriers arising from removal of the hydration shell, formation of highly curved structures, and, eventually, fusion pore widening. Here, we measured the force-dependent lifetime of fusion intermediates using membrane-coated silica spheres attached to cantilevers of an atomic-force microscope. Analysis of time traces obtained from force-clamp experiments allowed us to unequivocally assign steps in deflection of the cantilever to membrane states during the SNARE-mediated fusion with solid-supported lipid bilayers. Read More

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November 2016

Regulation of actin catch-slip bonds with a RhoA-formin module.

Sci Rep 2016 10 12;6:35058. Epub 2016 Oct 12.

Wallace H Coulter Department of Biomedical Engineering Georgia Institute of Technology and Emory University, Atlanta, GA, USA.

The dynamic turnover of the actin cytoskeleton is regulated cooperatively by force and biochemical signaling. We previously demonstrated that actin depolymerization under force is governed by catch-slip bonds mediated by force-induced K113:E195 salt-bridges. Yet, the biochemical regulation as well as the functional significance of actin catch bonds has not been elucidated. Read More

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October 2016

Molecular determinants of cadherin ideal bond formation: Conformation-dependent unbinding on a multidimensional landscape.

Proc Natl Acad Sci U S A 2016 09 12;113(39):E5711-20. Epub 2016 Sep 12.

Department of Physics and Astronomy, Iowa State University, Ames, IA 50011; Ames Laboratory, US Department of Energy, Ames, IA 50011;

Classical cadherin cell-cell adhesion proteins are essential for the formation and maintenance of tissue structures; their primary function is to physically couple neighboring cells and withstand mechanical force. Cadherins from opposing cells bind in two distinct trans conformations: strand-swap dimers and X-dimers. As cadherins convert between these conformations, they form ideal bonds (i. Read More

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September 2016

An Atomic Force Microscope with Dual Actuation Capability for Biomolecular Experiments.

Sci Rep 2016 06 7;6:27567. Epub 2016 Jun 7.

Department of Electrical and Electronics Engineering, Bogazici University, Bebek 34342 Istanbul, Turkey.

We report a modular atomic force microscope (AFM) design for biomolecular experiments. The AFM head uses readily available components and incorporates deflection-based optics and a piezotube-based cantilever actuator. Jetted-polymers have been used in the mechanical assembly, which allows rapid manufacturing. Read More

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Fully Atomistic Simulations of Protein Unfolding in Low Speed Atomic Force Microscope and Force Clamp Experiments with the Help of Boxed Molecular Dynamics.

J Phys Chem B 2016 Feb 25;120(4):700-8. Epub 2016 Jan 25.

School of Chemistry, University of Leeds , Leeds LS2 9JT, U.K.

The results of boxed dynamics (BXD) fully atomistic simulations of protein unfolding by atomic force microscopy (AFM) in both force clamp (FC) and velocity clamp (VC) modes are reported. In AFM experiments the unfolding occurs on a time scale which is too long for standard atomistic molecular dynamics (MD) simulations, which are usually performed with the addition of forces which exceed those of experiment by many orders of magnitude. BXD can reach the time scale of slow unfolding and sample the very high free energy unfolding pathway, reproducing the experimental dependence of pulling force against extension and extension against time. Read More

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February 2016

Direct Observation of the Dynamics of Self-Assembly of Individual Solvation Layers in Molecularly Confined Liquids.

Phys Rev Lett 2015 Jun 25;114(25):258303. Epub 2015 Jun 25.

Department of Physics and Randall Division of Cell and Molecular Biophysics, King's College London, Strand, WC2R 2LS London, United Kingdom.

Confined liquids organize in solidlike layers at the liquid-substrate interface. Here we use force-clamp spectroscopy AFM to capture the equilibrium dynamics between the broken and reformed states of an individual solvation layer in real time. Kinetic measurements demonstrate that the rupture of each individual solvation layer in structured liquids is driven by the rupture of a single interaction for 1-undecanol and by two interactions in the case of the ionic liquid ethylammonium nitrate. Read More

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Imaging viscoelastic properties of live cells by AFM: power-law rheology on the nanoscale.

Soft Matter 2015 Jun;11(23):4584-91

Institute of Applied Physics, University of Tübingen, Auf der Morgenstelle 10, 72076 Tübingen, Germany.

We developed force clamp force mapping (FCFM), an atomic force microscopy (AFM) technique for measuring the viscoelastic creep behavior of live cells with sub-micrometer spatial resolution. FCFM combines force-distance curves with an added force clamp phase during tip-sample contact. From the creep behavior measured during the force clamp phase, quantitative viscoelastic sample properties are extracted. Read More

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Catch bond interaction between cell-surface sulfatase Sulf1 and glycosaminoglycans.

Biophys J 2015 Apr;108(7):1709-1717

Experimental Biophysics, Physics Faculty, Bielefeld University, Bielefeld, Germany.

In biological adhesion, the biophysical mechanism of specific biomolecular interaction can be divided in slip and catch bonds, respectively. Conceptually, slip bonds exhibit a reduced bond lifetime under increased external force and catch bonds, in contrast, exhibit an increased lifetime (for a certain force interval). Since 2003, a handful of biological systems have been identified to display catch bond properties. Read More

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Mechanically induced silyl ester cleavage under acidic conditions investigated by AFM-based single-molecule force spectroscopy in the force-ramp mode.

Faraday Discuss 2014 ;170:357-67

Munich University of Applied Sciences, Department of Applied Natural Sciences and Mechatronics, Lothstr. 34, 80335 Munich, Germany.

AFM-based dynamic single-molecule force spectroscopy was used to stretch carboxymethylated amylose (CMA) polymers, which have been covalently tethered between a silanized glass substrate and a silanized AFM tip via acid-catalyzed ester condensation at pH 2.0. Rupture forces were measured as a function of temperature and force loading rate in the force-ramp mode. Read More

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Reading single DNA with DNA polymerase followed by atomic force microscopy.

J Am Chem Soc 2014 Oct 17;136(39):13754-60. Epub 2014 Sep 17.

School of Interdisciplinary Bioscience and Bioengineering, ‡Department of Chemistry, and §Department of Life Sciences, Pohang University of Science and Technology , San 31 Hyoja-dong, Pohang, 790-784, Korea.

The importance of DNA sequencing in the life sciences and personalized medicine is continually increasing. Single-molecule sequencing methods have been developed to analyze DNA directly without the need for amplification. Here, we present a new approach to sequencing single DNA molecules using atomic force microscopy (AFM). Read More

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October 2014

Altered thiol chemistry in human amyotrophic lateral sclerosis-linked mutants of superoxide dismutase 1.

J Biol Chem 2014 Sep 4;289(39):26722-26732. Epub 2014 Aug 4.

Department of Biological Sciences, Columbia University, New York, New York 10027, and; Vascular Biology and Inflammation Department, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Cl. Melchor Fernández Almagro 3, 28029 Madrid, Spain.

Neurodegenerative diseases share a common characteristic, the presence of intracellular or extracellular deposits of protein aggregates in nervous tissues. Amyotrophic Lateral Sclerosis (ALS) is a severe and fatal neurodegenerative disorder, which affects preferentially motoneurons. Changes in the redox state of superoxide dismutase 1 (SOD1) are associated with the onset and development of familial forms of ALS. Read More

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September 2014

Nanomechanics of HaloTag tethers.

J Am Chem Soc 2013 Aug 19;135(34):12762-71. Epub 2013 Aug 19.

Department of Biological Sciences, Columbia University, 1212 Amsterdam Avenue, New York, New York 10027, USA.

The active site of the Haloalkane Dehydrogenase (HaloTag) enzyme can be covalently attached to a chloroalkane ligand providing a mechanically strong tether, resistant to large pulling forces. Here we demonstrate the covalent tethering of protein L and I27 polyproteins between an atomic force microscopy (AFM) cantilever and a glass surface using HaloTag anchoring at one end and thiol chemistry at the other end. Covalent tethering is unambiguously confirmed by the observation of full length polyprotein unfolding, combined with high detachment forces that range up to ∼2000 pN. Read More

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Tracking unfolding and refolding reactions of single proteins using atomic force microscopy methods.

Methods 2013 Apr 20;60(2):151-60. Epub 2013 Mar 20.

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch at Galveston, TX 77555, USA.

During the last two decades single-molecule manipulation techniques such as atomic force microscopy (AFM) has risen to prominence through their unique capacity to provide fundamental information on the structure and function of biomolecules. Here we describe the use of single-molecule AFM to track protein unfolding and refolding pathways, enzymatic catalysis and the effects of osmolytes and chaperones on protein stability and folding. We will outline the principles of operation for two different AFM pulling techniques: length clamp and force-clamp and discuss prominent applications. Read More

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Single-molecule studies of disulfide bond reduction pathways used by human thioredoxin.

Biophys Chem 2013 Mar-Apr;173-174:31-8. Epub 2013 Jan 29.

Department of Physics, Kansas State University, 307 Cardwell Hall, Manhattan, KS, USA.

Disulfide bond reduction pathways used by human thioredoxin (hTrx) are studied at the single molecule level using a recombinant protein (I27SS)8. (I27SS)8 contains eight tandem repeats of identical immunoglobulin-like modules with one disulfide bond in each module. Single (I27SS)8 molecules are stretched at constant force applied by a cantilever in a force-clamp mode of atomic force microscopy (FC-AFM). Read More

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September 2013

Atomic force microscope based biomolecular force-clamp measurements using a micromachined electrostatic actuator.

Ultramicroscopy 2012 Nov 31;122:26-31. Epub 2012 Jul 31.

Department of Electrical and Electronics Engineering, Bogazici University, Bebek, TR-34342 Istanbul, Turkey.

The authors describe a method for biomolecular force clamp measurements using atomic force microscope (AFM) cantilevers and micromachined membrane-based electrostatic actuators. The actuators comprise of Parylene membranes with embedded side actuation electrodes and are fabricated on a silicon substrate. The devices have a displacement range of 1. Read More

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November 2012

Unraveling solvent-mediated reaction pathways leading to regiospecific mechanochemical cleavage of disulfide bonds in peptides.

Chemistry 2012 Jul 17. Epub 2012 Jul 17.

Lehrstuhl für Theoretische Chemie, Ruhr-Universität Bochum, 44780 Bochum (Germany).

Stressing disulfide bonds! Nucleophilic thiol-disulfide exchange reactions within the I27 domain of titin were previously investigated with force clamp AFM. Here, all possible pathways associated with disulfide bond scission at constant tensile force are revealed in terms of end-to-end distances by using force clamp molecular dynamics. The simulations, together with experimental data unravel the competition between mechanochemical bond activation and solvent-mediated regiospecificity exhibited during SS bond cleavage due to the nucleophilic substitution mechanism within a stretched peptide. Read More

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Single molecule force spectroscopy using polyproteins.

Chem Soc Rev 2012 Jul 30;41(14):4781-96. Epub 2012 May 30.

School of Physics and Astronomy, University of Leeds, Leeds LS2 9JT, United Kingdom.

In recent years single molecule force spectroscopy has emerged as a powerful new tool to explore the mechanical stability and folding pathways of individual proteins. This technique is used to apply a stretching force between two points of a protein, unfolding the protein to an extended state. By measuring the unfolding and folding trajectories of individual proteins, insight can be gained into the physical mechanisms of protein folding. Read More

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Complete noise analysis of a simple force spectroscopy AFM setup and its applications to study nanomechanics of mammalian Notch 1 protein.

Nanotechnology 2012 May 5;23(17):175101. Epub 2012 Apr 5.

Department of Physics, Kansas State University, Manhattan, KS 66506, USA.

We describe a complete noise analysis and application of a custom made AFM force spectroscopy setup on pulling a recombinant protein with an NRR domain of mouse Notch 1. Our table top AFM setup is affordable, has an open architecture, and is easily transferable to other laboratories. Its calculated noise characteristics are dominated by the Brownian noise with 2% non-Brownian components integrated over the first thermally induced resonance of a typical cantilever. Read More

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AFM-based force-clamp monitors lipid bilayer failure kinetics.

Langmuir 2012 Apr 5;28(15):6403-10. Epub 2012 Apr 5.

Institute for Bioengineering of Catalonia (IBEC), 15-21 Baldiri I Reixac, 08028 Barcelona, Spain.

The lipid bilayer rupture phenomenon is here explored by means of atomic force microscopy (AFM)-based force clamp, for the first time to our knowledge, to evaluate how lipid membranes respond when compressed under an external constant force, in the range of nanonewtons. Using this method, we were able to directly quantify the kinetics of the membrane rupture event and the associated energy barriers, for both single supported bilayers and multibilayers, in contradistinction to the classic studies performed at constant velocity. Moreover, the affected area of the membrane during the rupture process was calculated using an elastic deformation model. Read More

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Single-molecule force-clamp experiments reveal kinetics of mechanically activated silyl ester hydrolysis.

ACS Nano 2012 Feb 25;6(2):1314-21. Epub 2012 Jan 25.

Department of Precision- and Micro-Engineering, Engineering Physics, Munich University of Applied Sciences, Lothstr. 34, 80335 Munich, Germany.

We have investigated the strength of silyl ester bonds formed between carboxymethylated amylose (CMA) molecules and silane-functionalized silicon oxide surfaces using AFM-based single-molecule force spectroscopy in the force-clamp mode. Single tethered CMA molecules were picked up, and bond lifetimes were determined at constant clamp forces of 0.8, 1. Read More

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February 2012

DNA condensation by TmHU studied by optical tweezers, AFM and molecular dynamics simulations.

J Biol Phys 2011 Jan 9;37(1):117-31. Epub 2010 Oct 9.

The compaction of DNA by the HU protein from Thermotoga maritima (TmHU) is analysed on a single-molecule level by the usage of an optical tweezers-assisted force clamp. The condensation reaction is investigated at forces between 2 and 40 pN applied to the ends of the DNA as well as in dependence on the TmHU concentration. At 2 and 5 pN, the DNA compaction down to 30% of the initial end-to-end distance takes place in two regimes. Read More

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January 2011

Force-clamp measurements of receptor-ligand interactions.

Methods Mol Biol 2011 ;736:331-53

Miller School of Medicine, University of Miami, Miami, FL, USA.

Protein-protein interactions are the basis of both biochemical and biophysical signaling of living cells. In many cases, the receptor is present on the cell surface while the ligand is in solution or linked to another support (extracellular matrix or another cell). In the case of cellular adhesion, forces are continuously applied to receptor-ligand complexes and, as a consequence, the dissociation kinetics of the bonds may change. Read More

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October 2011