Single-molecule Force Microscopy Research Articles

Aptamer-based biotherapeutic conjugate for shear responsive release of Von Willebrand factor A1 domain.

Smart polymers that mimic and even surpass the functionality of natural responsive materials have been actively researched. This study explores the design and characterization of a Single-MOlecule-based material REsponsive to Shear (SMORES) for the targeted release of A1, the platelet binding domain of the blood clotting protein von Willebrand factor (VWF). Each SMORES construct employs an aptamer molecule as the flow transducer and a microparticle to sense and amplify the hydrodynamic force. Within the construct, the aptamer, ARC1172, undergoes conformational changes beyond a shear stress threshold, mimicking the shear-responsive behavior of VWF. This conformational alteration modulates the bioavailability of its target, the VWF-A1 domain, ultimately releasing it at elevated shear. Through optical tweezer-based single-molecule force measurement, ARC1172s role as a force transducer was assessed by examining its unfolding under constant pulling force. We also investigated its refolding rate as a function of force under varied relaxation periods. These analyses revealed a narrow range of threshold forces (3-7 pN) governing the transition between folded and unfolded states. We subsequently constructed the SMORES material by conjugating ARC1172 and a microbead, and immobilizing the other end of the aptamer on a substrate. Single-molecule flow experiments on immobilized SMORES constructs revealed a peak A1 domain release within a flow rate range of (40-70 μL min-1). A COMSOL Multiphysics model translated these flow rates to total forces of 3.10 pN-5.63 pN experienced by the aptamers, aligning with single-molecule force microscopy predictions. Evaluation under variable flow conditions showed a peak binding of A1 to the platelet glycoprotein Ib (GPIB) within the same force range, confirming released payload functionality. Building on knowledge of aptamer biomechanics, this study presents a new strategy to create shear-stimulated biomaterials based on single biomolecules.

Molecular Recognition in Confined Space Elucidated with DNA Nanopores and Single-Molecule Force Microscopy.

The binding of ligands to receptors within a nanoscale small space is relevant in biology, biosensing, and affinity filtration. Binding in confinement can be studied with biological systems but under the limitation that essential parameters cannot be easily controlled including receptor type and position within the confinement and its dimensions. Here we study molecular recognition with a synthetic confined nanopore with controllable pore dimension and molecular DNA receptors at different depth positions within the channel. Binding of a complementary DNA strand is studied at the single-molecule level with atomic force microscopy. Following the analysis, kinetic association rates are lower for receptors positioned deeper inside the pore lumen while dissociation is faster and requires less force. The phenomena are explained by the steric constraints on molecular interactions in confinement. Our study is the first to explore recognition in DNA nanostructures with atomic force microscopy and lays out new tools to further quantify the effect of nanoconfinement on molecular interactions.

Molecular Dynamics-Assisted Interpretation of Experimentally Determined Intrinsically Disordered Protein Conformational Components: The Case of Human α-Synuclein.

Mass spectrometry and single molecule force microscopy are two experimental approaches able to provide structural information on intrinsically disordered proteins (IDPs). These techniques allow the dissection of conformational ensembles in their main components, although at a low-resolution level. In this work, we interpret the results emerging from these experimental approaches on human alpha synuclein (AS) by analyzing a previously published 73 μs-long molecular-dynamics (MD) simulation of the protein in explicit solvent. We further compare MD-based predictions of single molecule Förster resonance energy transfer (smFRET) data of AS in solution with experimental data. The combined theoretical and experimental data provide a description of AS main conformational ensemble, shedding light into its intramolecular interactions and overall structural compactness. This analysis could be easily transferred to other IDPs.

N501Y mutation of spike protein in SARS-CoV-2 strengthens its binding to receptor ACE2.

SARS-CoV-2 has been spreading around the world for the past year. Recently, several variants such as B.1.1.7 (alpha), B.1.351 (beta), and P.1 (gamma), which share a key mutation N501Y on the receptor-binding domain (RBD), appear to be more infectious to humans. To understand the underlying mechanism, we used a cell surface-binding assay, a kinetics study, a single-molecule technique, and a computational method to investigate the interaction between these RBD (mutations) and ACE2. Remarkably, RBD with the N501Y mutation exhibited a considerably stronger interaction, with a faster association rate and a slower dissociation rate. Atomic force microscopy (AFM)-based single-molecule force microscopy (SMFS) consistently quantified the interaction strength of RBD with the mutation as having increased binding probability and requiring increased unbinding force. Molecular dynamics simulations of RBD-ACE2 complexes indicated that the N501Y mutation introduced additional π-π and π-cation interactions that could explain the changes observed by force microscopy. Taken together, these results suggest that the reinforced RBD-ACE2 interaction that results from the N501Y mutation in the RBD should play an essential role in the higher rate of transmission of SARS-CoV-2 variants, and that future mutations in the RBD of the virus should be under surveillance.

Probing fibronectin adsorption on chemically defined surfaces by means of single molecule force microscopy

Atomic force microscope (AFM) based single molecule force spectroscopy (SMFS) and a quartz crystal microbalance (QCM) were respectively employed to probe interfacial characteristics of fibronectin fragment FNIII8–14 and full-length fibronectin (FN) on CH3–, OH–, COOH–, and NH2-terminated alkane-thiol self-assembled monolayers (SAMs). Force-distance curves acquired between hexahistidine-tagged FNIII8–14 immobilised on trisNTA-Ni2+ functionalized AFM cantilevers and the OH and COOH SAM surfaces were predominantly ‘loop-like’ (76% and 94% respectively), suggesting domain unfolding and preference for ‘end-on’ oriented binding, while those generated with NH2 and CH3 SAMs were largely ‘mixed type’ (81% and 86%, respectively) commensurate with unravelling and desorption, and ‘side-on’ binding. Time-dependent binding of FN to SAM-coated QCM crystals occurred in at least two phases: initial rapid coverage over the first 5 min; and variably diminishing adsorption thereafter (5–70 min). Loading profiles and the final hydrated surface concentrations reached (~ 950, ~ 1200, ~ 1400, ~ 1500 ng cm−2 for CH3, OH, COOH and NH2 SAMs) were consistent with: space-filling ‘side-on’ orientation and unfolding on CH3 SAM; greater numbers of FN molecules arranged ‘end-on’ on OH and especially COOH SAMs; and initial ‘side-on’ contact, followed by either (1) gradual tilting to a space-saving ‘end-on’ configuration, or (2) bi-/multi-layer adsorption on NH2 SAM.

A pathway for helical membrane proteins

Protein Folding Membrane proteins are inserted into cell membranes while they are being translated and may fold concurrently into their secondary and tertiary structures. Choi et al. describe a single-molecule force microscopy technique that allowed them to monitor folding of helical membrane proteins in vesicles and bicelles. Two helical membrane proteins, the Escherichia coli rhomboid protease GlpG and the human β2-adrenergic receptor, both folded from the N to the C terminus, with structures forming in units of helical hairpins. In the cell, this would allow these proteins to begin folding while being translated. Science , this issue p. [1150][1] [1]: /lookup/doi/10.1126/science.aaw8208

Watching helical membrane proteins fold reveals a common N-to-C-terminal folding pathway.

To understand membrane protein biogenesis, we need to explore folding within a bilayer context. Here, we describe a single-molecule force microscopy technique that monitors the folding of helical membrane proteins in vesicle and bicelle environments. After completely unfolding the protein at high force, we lower the force to initiate folding while transmembrane helices are aligned in a zigzag manner within the bilayer, thereby imposing minimal constraints on folding. We used the approach to characterize the folding pathways of the Escherichia coli rhomboid protease GlpG and the human β2-adrenergic receptor. Despite their evolutionary distance, both proteins fold in a strict N-to-C-terminal fashion, accruing structures in units of helical hairpins. These common features suggest that integral helical membrane proteins have evolved to maximize their fitness with cotranslational folding.

Reversible Unfolding and Folding of the Metalloprotein Ferredoxin Revealed by Single-Molecule Atomic Force Microscopy.

Plant type [2Fe-2S] ferredoxins function primarily as electron transfer proteins in photosynthesis. Studying the unfolding-folding of ferredoxins in vitro is challenging, because the unfolding of ferredoxin is often irreversible due to the loss or disintegration of the iron-sulfur cluster. Additionally, the in vivo folding of holo-ferredoxin requires ferredoxin biogenesis proteins. Here, we employed atomic force microscopy-based single-molecule force microscopy and protein engineering techniques to directly study the mechanical unfolding and refolding of a plant type [2Fe-2S] ferredoxin from cyanobacteria Anabaena. Our results indicate that upon stretching, ferredoxin unfolds in a three-state mechanism. The first step is the unfolding of the protein sequence that is outside and not sequestered by the [2Fe-2S] center, and the second one relates to the force-induced rupture of the [2Fe-2S] metal center and subsequent unraveling of the protein structure shielded by the [2Fe-2S] center. During repeated stretching and relaxation of a single polyprotein, we observed that the completely unfolded ferredoxin can refold to its native holo-form with a fully reconstituted [2Fe-2S] center. These results demonstrate that the unfolding-refolding of individual ferredoxin is reversible at the single-molecule level, enabling new avenues of studying both folding-unfolding mechanisms, as well as the reactivity of the metal center of metalloproteins in vitro.

Conductive Polymer Binder for High-Tap-Density Nanosilicon Material for Lithium-Ion Battery Negative Electrode Application.

High-tap-density silicon nanomaterials are highly desirable as anodes for lithium ion batteries, due to their small surface area and minimum first-cycle loss. However, this material poses formidable challenges to polymeric binder design. Binders adhere on to the small surface area to sustain the drastic volume changes during cycling; also the low porosities and small pore size resulting from this material are detrimental to lithium ion transport. This study introduces a new binder, poly(1-pyrenemethyl methacrylate-co-methacrylic acid) (PPyMAA), for a high-tap-density nanosilicon electrode cycled in a stable manner with a first cycle efficiency of 82%-a value that is further improved to 87% when combined with graphite material. Incorporating the MAA acid functionalities does not change the lowest unoccupied molecular orbital (LUMO) features or lower the adhesion performance of the PPy homopolymer. Our single-molecule force microscopy measurement of PPyMAA reveals similar adhesion strength between polymer binder and anode surface when compared with conventional polymer such as homopolyacrylic acid (PAA), while being electronically conductive. The combined conductivity and adhesion afforded by the MAA and pyrene copolymer results in good cycling performance for the high-tap-density Si electrode.

May the Force Be With You: Unfolding Lipid-Protein Interactions By Single-Molecule Force Spectroscopy

In this issue of Structure, Serdiuk etal. report the use of single-molecule force microscopy to establish a role for phosphatidylethanolamine in promoting the native fold of lactose permease, thereby preventing it from populating a functionally defective, nonnative conformation (Serdiuk etal., 2015).

Action of the Hsp70 chaperone system observed with single proteins.

In Escherichia coli, the binding of non-native protein substrates to the Hsp70 chaperone DnaK is mediated by the co-chaperone DnaJ. DnaJ accelerates ATP hydrolysis on DnaK, by closing the peptide-binding cleft of DnaK. GrpE catalysed nucleotide exchange and ATP re-binding then lead to substrate release from DnaK, allowing folding. Here we refold immunoglobulin 27 (I27) to better understand how DnaJ-DnaK-GrpE chaperones cooperate. When DnaJ is present, I27 is less likely to misfold and more likely to fold, whereas the unfolded state remains unaffected. Thus, the 'holdase' DnaJ shows foldase behaviour. Misfolding of I27 is fully abrogated when DnaJ cooperates with DnaK, which stabilizes the unfolded state and increases the probability of folding. Addition of GrpE shifts the unfolded fraction of I27 to pre-chaperone levels. These insights reveal synergistic mechanisms within the evolutionary highly conserved Hsp70 system that prevent substrates from misfolding and promote their productive transition to the native state.

Unravelling the Properties of Single α-Helical Domains in Myosin and other Proteins

In most proteins, α-helices are stabilised by interactions with neighbouring secondary structure elements (e.g. coiled-coils). However, we recently showed that many proteins contain single α-helical (SAH) domains, which are stable in isolation and commonly inserted between two different functional domains. SAH domains are rich in arginine, lysine and glutamic acid residues. Their stability arises from the many potential (i, i ± 4) and (i, i ± 3) intrahelical interactions between either R and E, or K and E.To date, the best-studied SAH domains are those from myosins 6 and 10, and the Dictyostelium myosin myoM, where they are likely to form part of the functional lever. A SAH domain is also predicted for myosin 7a. We have shown that the SAH domain can functionally substitute for the canonical lever in myosin 5a in vitro. In cells, myosin 10 missing its SAH domain still moves to the tips of filopodia but with a reduced velocity.We have now determined that the SAH domains unfold at forces of less than ∼50 pN using single molecule force microscopy. Moreover, SAH domains unfold non-co-operatively during thermal melting, and have a high propensity to refold completely when the temperature is returned back to 10 °C, measured by circular dichroism. Molecular simulations agree with all the experimental data available, confirming the surprising stability of SAH domains and revealing unique properties that set the SAH domains apart. Among these is the ability to recover helical conformation rapidly after being extended through application of force.Thus SAH domains appear to function as ‘stretchable’ elements, unfolding and rapidly refolding at relatively low forces. In myosins, this may enable these motors to hold on to and traffic their cargoes in the crowded environment of the cell.

Specialized whisker probes for nanodiagnostics

A short review of specialized probes for nanodiagnostics with an accent to using for this aim of silicon whisker tip probes prepared by crystal growing from vapor phase. The probes ensure significant advantages in comparison with the standard probes that are produced by etching single-crystalline silicon wafers. Whisker probes for chemical force microscopy and for biological force microscopy are considered; their development for new versions, “single-molecule force microscopy” has been noted. Possibilities of the whisker probes for magnetic force microscopy and its new version, so-called “nano cluster magnetic force microscopy” that can be useful at chemical design on nanosized high-spin molecules are analyzed. Possibilities of the whisker probes for improvement of resolution and sensitivity of the capacity force microscopy for investigations in impurity distribution in semiconductor crystals and films are considered.

Detection of metal binding sites on functional S-layer nanoarrays using single molecule force spectroscopy

Crystalline bacterial cell surface layers (S-layers) show the ability to recrystallize into highly regular pattern on solid supports. In this study, the genetically modified S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177, carrying a hexa-histidine tag (His(6)-tag) at the C-terminus, was used to generate functionalized two-dimensional nanoarrays on a silicon surface. Atomic force microscopy (AFM) was applied to explore the topography and the functionality of the fused His(6)-tags. The accessibility of the His(6)-tags was demonstrated by in-situ anti-His-tag antibody binding to the functional S-layer array. The metal binding properties of the His(6)-tag was investigated by single molecule force microscopy. For this purpose, newly developed tris-NTA was tethered to the AFM tips via a flexible polyethylene glycol (PEG) linker. The functionalized tips showed specific interactions with S-layer containing His(6)-tags in the presence of nickel ions. Thus the His(6)-tag is located at the outer surface of the S-layer and can be used for stable but reversible attachment of functional tris-NTA derivatives.

Single Molecule Force Microscopy on Cells and Biological Membranes

Atomic force microscopy (AFM) enables high resolution topographic imaging of biological samples under near-physiological conditions. Therefore, the AFM is optimally suited for investigation of biological membranes and cell surfaces, as exemplified by studies on bacterial S-layers, purple membranes and cultured living cells. Topographic imaging allows visualizing single proteins and protein assemblies in native membranes, as well as substructures of live cells, such as cytoskeletal architecture. In addition to high-resolution imaging, the measurement of mechanical forces yields detailed insight into structure-function relationships of molecular processes in their native environment. In molecular recognition force microscopy, interaction forces between tip-bound ligands and membrane-embedded receptors can be studied under well-controlled buffer conditions and effectors concentrations. In case of low lateral density and inhomogeneous distribution of the target molecules in a cell membrane, fluorescence microscopy can help to guide the AFM tip to the membrane proteins of interest, which can subsequently be investigated by molecular recognition force microscopy. Keywords: Atomic force microscopy (AFM), Force spectroscopy, Molecular recognition, Recognition imaging, Molecular forces, Cell membranes, Membrane proteins

Noncovalent Cross-Linking of Casein by Epigallocatechin Gallate Characterized by Single Molecule Force Microscopy

Interaction of the tea polyphenol epigallocatechin gallate (EGCG) with beta-casein in milk affects the taste of tea and also affects the stability of the tea and the antioxidant ability of the EGCG. In addition, interaction of polyphenols with the chemically similar salivary proline-rich proteins is largely responsible for the astringency of tea and red wine. With the use of single molecule force microscopy, we demonstrate that the interaction of EGCG with a single casein molecule is multivalent and leads to reduction in the persistence length of casein as calculated using the wormlike chain model and a reduction in its radius of gyration. The extra force required to stretch casein in the presence of EGCG is largely entropic, suggesting that multivalent hydrophobic interactions cause a compaction of the casein micelle.

Single molecule study of binding force between transcription factorTINY and its DNA responsive element

The interaction force between transcription factor TINY and its corresponding cis-element has been studied with single molecule force microscopy. TINY belongs to the AP2/EREBP family, which is the second largest transcription factor family in plant and plays an important role in the genetic regulation of plant development and growth. The results showed that TINY bound to dehydration-responsive element (DRE) with the core sequence A/GCCGAC efficiently. The single molecule forces between TINY and DRE A/GCCGAC measured by atomic force microscopy were 83.5±3.4 and 81.4±4.9pN, respectively. Either the single base substitution of the DRE core sequence or point mutation of the key amino acid in TINY DNA-binding domain greatly reduced the binding strength. We have previously reported the method of using AFM to measure the interaction force between transcription factor ZmDREB1A, another AP2/EREBP family protein, and DRE responsive element. The results in this work further confirm the applicability and high sensitivity of AFM measurements in transcription factor–DNA interaction study. More interesting, it was found that 19th amino acid mutation of TINY resulted in the decrease of the binding force but the binding probability was the same as the wild type TINY. The reduced binding force was correlated with the reduced activity of TINY regulated reporter gene in the yeast one-hybrid experiment. It is thus expected that the single molecule force measurement offers valuable information on the analysis of transcription factor regulated gene expression.

Investigation of perylene photonic wires by combined single-molecule fluorescence and atomic force microscopy.

Multichromophoric systems can exhibit interesting optical and electronic properties due to excitonic interactions between dye units that are near to each other. An exquisite example found in nature is the light-harvesting antenna system in photosynthetic bacteria, which allow for efficient light absorption and unidirectional transfer of energy to the reaction center during photosynthesis.The design of artificial multichromophoric arrays that mimic these properties is a great challenge, the ultimate goal being the construction of molecular photonic and electronic devices. A unique insight into the physical behavior of multichromophoric systems is obtained with the help of single-molecule-detection techniques. Single-molecule force microscopy not only provides information on the dimensions of the aggregates but also on the internal organization of the chromophores, a crucial issue that governs their excitonic behavior. Single-molecule optical spectroscopy reveals characteristic features arising from exciton coupling, such as single-emitter behavior and collective fluorescent/ nonfluorescent states, properties that otherwise remain hidden when probing the average behavior of ensembles of molecules. In this work we have combined force and optical microscopy to investigate at the single-molecule level the properties of a new class of rigid multichromophoric polymers intended to act as synthetic antennas.

Single-Molecule Force Microscopy of Circularly Permuted Green Fluorescent Protein

We introduced new termini on surface loops of green fluorescent protein (GFP) by linking the original ones. The mechanical properties of two circularly permuted GFPs (cpGFPs) were examined by atomic force microscopy (AFM) and compared with those of the base GFPs. The unfolding results revealed different levels of the reduced mechanical stability of cpGFPs, and these levels were related to the proximity of the newly introduced termini to the central `β-can'.

The Force-Driven Conformations of Heparin Studied with Single Molecule Force Microscopy

Using single molecule force spectroscopy we examine the response of heparin chains to mechanical stretching. We find that at forces below 200pN heparin behaves as a simple entropic spring. At ∼200pN heparin displays a large enthalpic elasticity, which is evident as a pronounced plateau in the force-extension relationship. We determine that this enthalpic elasticity is produced by sugar rings of heparin flipping to more energetic and more extended conformations. We estimate that in vivo, the forces which stretch heparin are comparable to the forces that trigger conformational transitions in our single molecule atomic force microscopy measurements. We hypothesize that these conformational transitions have biological significance in that they provide a mechanism to finely regulate the affinity of various ligands toward heparin, for example, in secretory granules undergoing exocytosis and during the mechanical interactions between cells and the extracellular matrix.