Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Control of Ligand-Binding Specificity Using Photocleavable Linkers in AFM Force Spectroscopy.

ABSTRACT: In recent decades, atomic force microscopy (AFM), in particular the force spectroscopy mode, has become a method of choice to study biomolecular interactions at the single-molecule level. However, grafting procedures as well as determining binding specificity remain challenging. We report here an innovative approach based on a photocleavable group that enables in situ release of the ligands bound to the AFM tip and thus allows direct assessment of the binding specificity. Applicable to a wide variety of molecules, the strategy presented here provides new opportunities to study specific interactions and deliver single molecules with high spatiotemporal resolution in a wide range of applications, including AFM-based cell biology.

SUBMITTER: Koehler M

PROVIDER: S-EPMC7252943 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Control of Ligand-Binding Specificity Using Photocleavable Linkers in AFM Force Spectroscopy.

Koehler Melanie M Lo Giudice Cristina C Vogl Philipp P Ebner Andreas A Hinterdorfer Peter P Gruber Hermann J HJ Alsteens David D

Nano letters 20200427 5

In recent decades, atomic force microscopy (AFM), in particular the force spectroscopy mode, has become a method of choice to study biomolecular interactions at the single-molecule level. However, grafting procedures as well as determining binding specificity remain challenging. We report here an innovative approach based on a photocleavable group that enables in situ release of the ligands bound to the AFM tip and thus allows direct assessment of the binding specificity. Applicable to a wide va ...[more]

PMID: 32320256

Similar Datasets

Novel polymer linkers for single molecule AFM force spectroscopy.

Project description:Flexible polymer linkers play an important role in various imaging and probing techniques that require surface immobilization, including atomic force microscopy (AFM). In AFM force spectroscopy, polymer linkers are necessary for the covalent attachment of molecules of interest to the AFM tip and the surface. The polymer linkers tether the molecules and provide their proper orientation in probing experiments. Additionally, the linkers separate specific interactions from nonspecific short-range adhesion and serve as a reference point for the quantitative analysis of single molecule probing events. In this report, we present our results on the synthesis and testing of a novel polymer linker and the identification of a number of potential applications for its use in AFM force spectroscopy experiments. The synthesis of the linker is based on the well-developed phosphoramidate (PA) chemistry that allows the routine synthesis of linkers with predetermined lengths and PA composition. These linkers are homogeneous in length and can be terminated with various functional groups. PA linkers with different functional groups were synthesized and tested in experimental systems utilizing different immobilization chemistries. We probed interactions between complementary DNA oligonucleotides; DNA and protein complexes formed by the site-specific binding protein SfiI; and interactions between amyloid peptide (Aβ42). The results of the AFM force spectroscopy experiments validated the feasibility of the proposed approach for the linker design and synthesis. Furthermore, the properties of the tether (length, functional groups) can be adjusted to meet the specific requirements for different force spectroscopy experiments and system characteristics, suggesting that it could be used for a large number of various applications.

| S-EPMC3698867 | biostudies-literature

Ligand Binding Stabilizes Cellulosomal Cohesins as Revealed by AFM-based Single-Molecule Force Spectroscopy.

Project description:The cohesin-dockerin receptor-ligand family is the key element in the formation of multi-enzyme lignocellulose-digesting extracellular complexes called cellulosomes. Changes in a receptor protein upon binding of a ligand - commonly referred to as allostery - are not just essential for signalling, but may also alter the overall mechanical stability of a protein receptor. Here, we measured the change in mechanical stability of a library of cohesin receptor domains upon binding of their dockerin ligands in a multiplexed atomic force microscopy-based single-molecule force spectroscopy experiment. A parallelized, cell-free protein expression and immobilization protocol enables rapid mechanical phenotyping of an entire library of constructs with a single cantilever and thus ensures high throughput and precision. Our results show that dockerin binding increases the mechanical stability of every probed cohesin independently of its original folding strength. Furthermore, our results indicate that certain cohesins undergo a transition from a multitude of different folds or unfolding pathways to a single stable fold upon binding their ligand.

| S-EPMC6018229 | biostudies-literature

Mechanism of amyloid ?-protein dimerization determined using single-molecule AFM force spectroscopy.

Project description:A?42 and A?40 are the two primary alloforms of human amyloid ?-protein (A?). The two additional C-terminal residues of A?42 result in elevated neurotoxicity compared with A?40, but the molecular mechanism underlying this effect remains unclear. Here, we used single-molecule force microscopy to characterize interpeptide interactions for A?42 and A?40 and corresponding mutants. We discovered a dramatic difference in the interaction patterns of A?42 and A?40 monomers within dimers. Although the sequence difference between the two peptides is at the C-termini, the N-terminal segment plays a key role in the peptide interaction in the dimers. This is an unexpected finding as N-terminal was considered as disordered segment with no effect on the A? peptide aggregation. These novel properties of A? proteins suggests that the stabilization of N-terminal interactions is a switch in redirecting of amyloids form the neurotoxic aggregation pathway, opening a novel avenue for the disease preventions and treatments.

| S-EPMC3791449 | biostudies-literature

Unequivocal single-molecule force spectroscopy of proteins by AFM using pFS vectors.

Project description:Nanomechanical analysis of proteins by single-molecule force spectroscopy based on atomic force microscopy is increasingly being used to investigate the inner workings of mechanical proteins and substrate proteins of unfoldase machines as well as to gain new insight into the process of protein folding. However, such studies are hindered by a number of technical problems, including the noise of the proximal region, ambiguous single-molecule identification, as well as difficulties in protein expression/folding and full-length purification. To overcome these major drawbacks in protein nanomechanics, we designed a family of cloning/expression vectors, termed pFS (plasmid for force spectroscopy), that essentially has an unstructured region to surmount the noisy proximal region, a homomeric polyprotein marker, a carrier to mechanically protect the protein of interest (only the pFS-2 version) that also acts as a reporter, and two purification tags. pFS-2 enables the unambiguous analysis of proteins with low mechanical stability or/and complex force spectra, such as the increasingly abundant class of intrinsically disordered proteins, which are hard to characterize by traditional bulk techniques and have important biological and clinical implications. The advantages, applications, and potential of this ready-to-go system are illustrated through the analysis of representative proteins.

| S-EPMC3274811 | biostudies-literature

Structural-mechanical characterization of nanoparticle exosomes in human saliva, using correlative AFM, FESEM, and force spectroscopy.

Project description:All living systems contain naturally occurring nanoparticles with unique structural, biochemical, and mechanical characteristics. Specifically, human saliva exosomes secreted by normal cells into saliva via exocytosis are novel biomarkers showing tumor-antigen enrichment during oral cancer. Here we show the substructure of single human saliva exosomes, using a new ultrasensitive low force atomic force microscopy (AFM) exhibiting substructural organization unresolvable in electron microscopy. We correlate the data with field emission scanning electron microscopy (FESEM) and AFM images to interpret the nanoscale structures of exosomes under varying forces. Single exosomes reveal reversible mechanical deformation displaying distinct elastic, 70-100 nm trilobed membrane with substructures carrying specific transmembrane receptors. Further, we imaged and investigated, using force spectroscopy with antiCD63 IgG functionalized AFM tips, highly specific and sensitive detection of antigenCD63, potentially useful cancer markers on individual exosomes. The quantitative nanoscale morphological, biomechanical, and surface biomolecular properties of single saliva exosomes are critical for the applications of exosomes for cancer diagnosis and as a model for developing new cell delivery systems.

| S-EPMC2866049 | biostudies-literature

Bulk chemical composition contrast from attractive forces in AFM force spectroscopy.

Project description:A key application of atomic force microscopy (AFM) is the measurement of physical properties at sub-micrometer resolution. Methods such as force-distance curves (FDCs) or dynamic variants (such as intermodulation AFM (ImAFM)) are able to measure mechanical properties (such as the local stiffness, k r) of nanoscopic heterogeneous materials. For a complete structure-property correlation, these mechanical measurements are considered to lack the ability to identify the chemical structure of the materials. In this study, the measured attractive force, F attr, acting between the AFM tip and the sample is shown to be an independent measurement for the local chemical composition and hence a complete structure-property correlation can be obtained. A proof of concept is provided by two model samples comprised of (1) epoxy/polycarbonate and (2) epoxy/boehmite. The preparation of the model samples allowed for the assignment of material phases based on AFM topography. Additional chemical characterization on the nanoscale is performed by an AFM/infrared-spectroscopy hybrid method. Mechanical properties (k r) and attractive forces (F attr) are calculated and a structure-property correlation is obtained by a manual principle component analysis (mPCA) from a k r/F attr diagram. A third sample comprised of (3) epoxy/polycarbonate/boehmite is measured by ImAFM. The measurement of a 2 × 2 µm cross section yields 128 × 128 force curves which are successfully evaluated by a k r/F attr diagram and the nanoscopic heterogeneity of the sample is determined.

| S-EPMC7849247 | biostudies-literature

Probing Interactions within the synaptic DNA-SfiI complex by AFM force spectroscopy.

Project description:SfiI belongs to a family of restriction enzymes that function as tetramers, binding two recognition regions for the DNA cleavage reaction. The SfiI protein is an attractive and convenient model for studying synaptic complexes between DNA and proteins capable of site-specific binding. The enzymatic action of SfiI has been very well characterized. However, the properties of the complex before the cleavage reaction are not clear. We used single-molecule force spectroscopy to analyze the strength of interactions within the SfiI-DNA complex. In these experiments, the stability of the synaptic complex formed by the enzyme and two DNA duplexes was probed in a series of approach-retraction cycles. In order to do this, one duplex was tethered to the surface and the other was tethered to the probe. The complex was formed by the protein present in the solution. An alternative setup, in which the protein was anchored to the surface, allowed us to probe the stability of the complex formed with only one duplex in the approach-retraction experiments, with the duplex immobilized at the probe tip. Both types of complexes are characterized by similar rupture forces. The stability of the complex was determined by measuring the dependence of rupture forces on force loading rates (dynamic force spectroscopy) and the results suggest that the dissociation reaction of the SfiI-DNA complex has a single energy barrier along the dissociation path. Dynamic force spectroscopy was instrumental in revealing the role of the 5 bp spacer region within the palindromic recognition site on DNA-SfiI in the stability of the complex. The data show that, although the change of non-specific sequence does not alter the position of the activation barrier, it changes values of the off rates significantly.

| S-EPMC1847770 | biostudies-literature

Accurate, explicit formulae for higher harmonic force spectroscopy by frequency modulation-AFM.

Project description:The nonlinear interaction between an AFM tip and a sample gives rise to oscillations of the cantilever at integral multiples (harmonics) of the fundamental resonance frequency. The higher order harmonics have long been recognized to hold invaluable information on short range interactions but their utilization has thus far been relatively limited due to theoretical and experimental complexities. In particular, existing approximations of the interaction force in terms of higher harmonic amplitudes generally require simultaneous measurements of multiple harmonics to achieve satisfactory accuracy. In the present letter we address the mathematical challenge and derive accurate, explicit formulae for both conservative and dissipative forces in terms of an arbitrary single harmonic. Additionally, we show that in frequency modulation-AFM (FM-AFM) each harmonic carries complete information on the force, obviating the need for multi-harmonic analysis. Finally, we show that higher harmonics may indeed be used to reconstruct short range forces more accurately than the fundamental harmonic when the oscillation amplitude is small compared with the interaction range.

| S-EPMC4311655 | biostudies-literature

Oriented Membrane Protein Reconstitution into Tethered Lipid Membranes for AFM Force Spectroscopy.

Project description:Membrane proteins act as a central interface between the extracellular environment and the intracellular response and as such represent one of the most important classes of drug targets. The characterization of the molecular properties of integral membrane proteins, such as topology and interdomain interaction, is key to a fundamental understanding of their function. Atomic force microscopy (AFM) and force spectroscopy have the intrinsic capabilities of investigating these properties in a near-native setting. However, atomic force spectroscopy of membrane proteins is traditionally carried out in a crystalline setup. Alternatively, model membrane systems, such as tethered bilayer membranes, have been developed for surface-dependent techniques. While these setups can provide a more native environment, data analysis may be complicated by the normally found statistical orientation of the reconstituted protein in the model membrane. We have developed a model membrane system that enables the study of membrane proteins in a defined orientation by single-molecule force spectroscopy. Our approach is demonstrated using cell-free expressed bacteriorhodopsin coupled to a quartz glass surface in a defined orientation through a protein anchor and reconstituted inside an artificial membrane system. This approach offers an effective way to study membrane proteins in a planar lipid bilayer. It can be easily transferred to all membrane proteins that possess a suitable tag and can be reconstituted into a lipid bilayer. In this respect, we anticipate that this technique may contribute important information on structure, topology, and intra- and intermolecular interactions of other seven-transmembrane helical receptors.

| S-EPMC5103026 | biostudies-literature

Nanopore force spectroscopy of aptamer-ligand complexes.

Project description:The stability of aptamer-ligand complexes is probed in nanopore-based dynamic force spectroscopy experiments. Specifically, the ATP-binding aptamer is investigated using a backward translocation technique, in which the molecules are initially pulled through an α-hemolysin nanopore from the cis to the trans side of a lipid bilayer membrane, allowed to refold and interact with their target, and then translocated back in the trans-cis direction. From these experiments, the distribution of bound and unbound complexes is determined, which in turn allows determination of the dissociation constant Kd ≈ 0.1 mM of the aptamer and of voltage-dependent unfolding rates. The experiments also reveal differences in binding of the aptamer to AMP, ADP, or ATP ligands. Investigation of an aptamer variant with a stabilized ATP-binding site indicates fast conformational switching of the original aptamer before ATP binding. Nanopore force spectroscopy is also used to study binding of the thrombin-binding aptamer to its target. To detect aptamer-target interactions in this case, the stability of the ligand-free aptamer-containing G-quadruplexes-is tuned via the potassium content of the buffer. Although the presence of thrombin was detected, limitations of the method for aptamers with strong secondary structures and complexes with nanomolar Kd were identified.

| S-EPMC3762367 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data