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In neuroscience, understanding the mechanics of synapses, especially the function of force-sensitive proteins at the molecular level, is essential. This need emphasizes the importance of precise measurement of synaptic protein interactions. Addressing this, we introduce high-resolution magnetic tweezers (MT) as a novel method to probe the mechanics of synapse-related proteins with high precision. We demonstrate this technique through studying SNARE-complexin interactions, crucial for synaptic transmission, showcasing its capability to apply specific forces to individual molecules. Our results reveal that high-resolution MT provides in-depth insights into the stability and dynamic transitions of synaptic protein complexes. This method is a significant advancement in synapse biology, offering a new tool for researchers to investigate the impact of mechanical forces on synaptic functions and their implications for neurological disorders.

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Static magnetic fields (SMFs) exhibit numerous biological effects and regulate the proliferation and differentiation of several adult stem cells. However, the role of SMFs in the self-renewal maintenance and developmental potential of pluripotent embryonic stem cells (ESCs) remains largely uninvestigated. Here, we show that SMFs promote the expression of the core pluripotent markers Sox2 and SSEA-1. Furthermore, SMFs facilitate the differentiation of ESCs into cardiomyocytes and skeletal muscle cells. Consistently, transcriptome analysis reveals that muscle lineage differentiation and skeletal system specification of ESCs are remarkably strengthened by SMF stimuli. Additionally, when treated with SMFs, C2C12 myoblasts exhibit an increased proliferation rate, improved expression of skeletal muscle markers and elevated myogenic differentiation capacity compared with control cells. Together, our data show that SMFs effectively promote muscle cell generation from pluripotent stem cells and myoblasts. The noninvasive and convenient physical stimuli can be used to increase the production of muscle cells in regenerative medicine and the manufacture of cultured meat in cellular agriculture.

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Monitoring the dimerization state of the mesenchymal-epithelial transition factor (Met) was essential for in-depth understanding of the tumor signal transduction network. At present, the dimerization activation pathway of Met protein was mainly studied at the macro level, while the research at the single molecule level was far from comprehensive. Herein, the dimerization activation of Met protein's extracellular domain induced by ligand hepatocyte growth factor (HGF) was dynamically studied by single-molecule force spectroscopy. Met protein was immobilized on a biomimetic lipid membrane for ensuring its physiological environment, and then the Met dimers were recognized by bivalent probe which was formed by two Met-binding aptamers. Then the dimeric state of Met protein could be distinguished from monomeric state of Met protein through some parameters, (such as unimodal ratio, bimodal ratio and separation work). The unimodal indicates the occurrence of single molecule binding event, and the bimodal represents the occurrence of double binding event (also represents the presence of Met dimer). Before HGF treatment, most of the Met protein on the lipid membrane was still in the form of monomer, so the unimodal ratio in the force curve was larger (78.8 ± 5.2%), and the bimodal ratio was smaller (17.0 ± 4.1%). After HGF treatment, the unimodal ratio decreased to 54.0 ± 7.4%, and the bimodal ratio increased to 43.2 ± 7.3%. It was due to the formation of dimers after the binding of Met protein on the fluidity lipid membrane with HGF. In addition, the average separation work increased to about 2 times after HGF treatment. Given that studies of Met protein dimerization inhibitors have contributed to the development of more potent and safe inhibitors to significantly inhibit tumor metastasis, the effects of different medicines (including anticoagulant medicines, different antibiotics and anti-cancer medicines) on the dimerization activation of Met protein were then explored by the platform described above. The results showed that anticoagulant medicines heparin and its analogs can significantly inhibit HGF-mediated Met protein activation, while different antibiotics and anticancer medicines had no significant effect on the dimerization of Met protein. This work provided a platform for studying protein dimerization as well as for screening Met protein dimerization inhibitors at the single-molecule level.

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Single-molecule force spectroscopy methods, such as AFM and magnetic tweezers, have proved extremely beneficial in elucidating folding pathways for soluble and membrane proteins. To identify factors that determine the force rupture levels in force-induced membrane protein unfolding, we applied our near-atomic-level Upside molecular dynamics package to study the vertical and lateral pulling of bacteriorhodopsin (bR) and GlpG, respectively. With our algorithm, we were able to selectively alter the magnitudes of individual interaction terms and identify that, for vertical pulling, hydrogen bond strength had the strongest effect, whereas other non-bonded protein and membrane-protein interactions had only moderate influences, except for the extraction of the last helix where the membrane-protein interactions had a stronger influence. The up-down topology of the transmembrane helices caused helices to be pulled out as pairs. The rate-limiting rupture event often was the loss of H-bonds and the ejection of the first helix, which then propagated tension to the second helix, which rapidly exited the bilayer. The pulling of the charged linkers across the membrane had minimal influence, as did changing the bilayer thickness. For the lateral pulling of GlpG, the rate-limiting rupture corresponded to the separation of the helices within the membrane, with the H-bonds generally being broken only afterward. Beyond providing a detailed picture of the rupture events, our study emphasizes that the pulling mode greatly affects the factors that determine the forces needed to unfold a membrane protein.

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The Staphylococcus aureus surface protein G (SasG) is associated with host colonisation and biofilm formation. As colonisation occurs at the liquid-substrate interface bacteria are subject to a myriad of external forces and, presumably as a consequence, SasG displays extreme mechanical strength. This mechanical phenotype arises from the B-domain; a repetitive region composed of alternating E and G5 subdomains. These subdomains have an unusual structure comprising collagen-like regions capped by triple-stranded ß-sheets. To identify the determinants of SasG mechanical strength, we characterised the mechanical phenotype and thermodynamic stability of 18 single substitution variants of a pseudo-wildtype protein. Visualising the mechanically-induced transition state at a residue-level by ϕ-value analysis reveals that the main force-bearing regions are the N- and C-terminal 'Mechanical Clamps' and their side-chain interactions. This is tailored by contacts at the pseudo-hydrophobic core interface. We also describe a novel mechanical motif - the collagen-like region and show that glycine to alanine substitutions, analogous to those found in Osteogenesis Imperfecta (brittle bone disease), result in a significantly reduced mechanical strength.

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PURPOSE: 9.4 T magnetic resonance imaging (MRI) has been initially tested on healthy human volunteers, but its future application will benefit more from experiments with animal disease models. In the meantime, high static magnetic fields (SMFs) have been shown to improve mice mental health and have anti-tumor potentials. METHODS: We compared the anti-tumor effects of 9.4 T SMF with or without a commonly used chemotherapy drug imatinib mesylate on BALB/c (Nu/Nu) mice bearing gastrointestinal stromal tumor GIST-T1 cells. The body weight, food/water consumption, complete blood count, blood biochemistry, tumor weight, HE and Ki67 stains were examined. Locomotor activity and cognitive functions were also measured by four behavior tests, including open field, elevated plus maze, three-chamber and tail suspension tests. RESULTS: We found that the tumor growth was inhibited up to 62.88% when treated with 9.4 T SMF alone for 200 h. More importantly, 9.4 T SMF combined with 20 mg/kg imatinib mesylate can result in 92.75% tumor suppression, which is close to the anti-tumor effect of high dose (80 mg/kg) imatinib. However, 80 mg/kg imatinib caused severe side effects, including significantly reduced gain of body weight, abnormal liver function and depressive behaviors in mice. In contrast, 9.4 T SMF treatment significantly reduced these side effects, especially for the depressive behaviors. CONCLUSION: Our results demonstrate that 9.4 T SMF not only has anti-tumor effects on its own, but also could improve the anti-tumor effect of imatinib mesylate, reduce its toxicity and improve the mice mental health, which unraveled the great clinical potentials of high SMF in future applications.

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PEGylation has been considered the gold standard method for the modification of various drug delivery systems since the last century. However, the impact of PEGylation on the dynamic interaction between drug carriers and cell membranes has not been quantitatively clarified. Herein, the cellular binding and receptor-mediated endocytosis of a model PEGylated polypeptide nanomicelle were systematically investigated at the single-particle level using AFM-based single-molecule force spectroscopy (SMFS) and force tracing. A self-assembled elastin-like polypeptide (ELP) nanomicelle, which is capable of cross-linking, gastrin-releasing peptide (GRP) modification, and PEGylation was prepared. The cross-linked ELP-based nanomicelles exhibited outstanding stability in a broad temperature range of 4-40 °C, which facilitate the drug loading, as well as our cell-nanomicelle study at the single particle level. The unbinding force between the cross-linked ELP-based nanomicelles and the GRP receptor (GRPR)-containing cell (PC-3) membranes was quantitatively measured by AFM-SMFS. It is found that the PEGylated GRP-displaying nanomicelles exhibit the highest unbinding force, indicating the enhanced specific binding effect of PEGylation. Furthermore, the receptor-mediated endocytosis of the cross-linked ELP-based nanomicelles was monitored with the help of force tracing based on AFM-SMFS. Our results show that PEGylation decreases the endocytic force, duration, and engulfment depth of the PEGylated GRP-displaying nanomicelles, but increases their endocytic velocity, which results from the elimination of non-specific interactions during endocytosis. These observations demonstrate the diverse and complex roles of PEGylation on the interaction of polypeptide nanomicelles to cell membranes and may shed light on the rational design of organic polymer-based drug delivery systems aiming for active and passive targeting strategies. STATEMENT OF SIGNIFICANCE: A self-assembled elastin-like polypeptide (ELP) nanomicelle, which can be easily cross-linked, gastrin-releasing peptide (GRP) modified, and PEGylated, is designed. The AFM-SMFS experiment shows that PEGylation can enhance specific binding of the nanomicelles to the receptors on cell membranes. The force tracing experiment indicates that PEGylation decreases the endocytic force as well as engulfment depth of the nanomicelles through the elimination of non-specific interactions. PEGylation can benefit the drug delivery systems aiming at active targeting, while might not be an ideal modification for drug carriers designed for passive targeting, whose cellular uptake mainly depends on non-specific interactions.

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Fundamental adsorption mechanisms of poly(acrylic acid) (PAA) electrolyte/oxide interfaces were analyzed by the combination of in-situ attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy and single molecule force spectroscopy (SMFS). The approach aims at a fundamental understanding of initial states of polymer fouling in chemical microreactors. While the presented FTIR-data provide information on adsorption and desorption kinetics, SMFS studies reveal the corresponding interfacial and intermolecular forces. Silicon oxide and oxide covered FeCr alloy films with small concentrations of Ni were chosen as reference systems for relevant technical reactor components. Adsorption and desorption studies were performed in aqueous electrolytes at acidic pH to simulate the polymerisation process. Ex-situ ellipsometry and atomic force microscopy (AFM) studies of the adsorbed polymer layers as well as X-ray photoelectron spectroscopy (XPS) of the oxide surfaces complemented the analytical approach. The comparison of the in-situ ATR spectroscopic results and the SMFS data revealed higher molecular adhesion forces on FeCr-oxide covered FeCr alloy films in comparison to the SiOx terminated surfaces. The different interactions could be assigned to the specific coordination bonds formed between the carboxylic acid group and surface metal ions in the case of FeCr alloy films. AFM images showed related changes in interfacial film formation.

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Human peroxiredoxin-5 (PRDX5) is a unique redox-sensitive protein that plays a dual role in brain ischemia-reperfusion injury. While intracellular PRDX5 has been reported to act as a neuroprotective antioxidative enzyme by scavenging peroxides, once released extracellularly from necrotic brain cells, the protein aggravates neural cell death by inducing expression of proinflammatory cytokines in macrophages through activation of Toll-like receptor (TLR) 2 (TLR2) and 4 (TLR4). Although recent evidence showed that PRDX5 was able to interact directly with TLR4, little is known regarding the role of the cysteine redox state of PRDX5 on its DAMP function. To gain insights into the role of PRDX5 redox-active cysteine residues in the TLR4-dependent proinflammatory activity of the protein, we used a recombinant human PRDX5 in the disulfide (oxidized) form and a mutant version lacking the peroxidatic cysteine, as well as chemically reduced and hyperoxidized PRDX5 proteins. We first analyzed the oxidation state and oligomerization profile by Western blot, mass spectrometry, and SEC-MALS. Using ELISA, we demonstrate that the disulfide bridge between the enzymatic cysteines is required to allow improved TLR4-dependent IL-8 secretion. Moreover, single-molecule force spectroscopy experiments revealed that TLR4 alone is not sufficient to discriminate the different PRDX5 redox forms. Finally, flow cytometry binding assays show that disulfide PRDX5 has a higher propensity to bind to the surface of living TLR4-expressing cells than the mutant protein. Taken together, these results demonstrate the importance of the redox state of PRDX5 cysteine residues on TLR4-induced inflammation.

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Hydrophobins are surface-active proteins produced by filamentous fungi. The amphiphilic structure of hydrophobins is very compact, containing a distinct hydrophobic patch on one side of the molecule, locked by four intramolecular disulfide bridges. Hydrophobins form dimers and multimers in solution to shield these hydrophobic patches from water exposure. Multimer formation in solution is dynamic, and hydrophobin monomers can be exchanged between multimers. Unlike class I hydrophobins, class II hydrophobins assemble into highly ordered films at the air-water interface. In order to increase our understanding of the strength and nature of the interaction between hydrophobins, we used atomic force microscopy for single molecule force spectroscopy to explore the molecular interaction forces between class II hydrophobins from Trichoderma reesei under different environmental conditions. A genetically engineered hydrophobin variant, NCys-HFBI, enabled covalent attachment of proteins to the apex of the atomic force microscopy cantilever tip and sample surfaces in controlled orientation with sufficient freedom of movement to measure molecular forces between hydrophobic patches. The measured rupture force between two assembled hydrophobins was ∼31 pN, at a loading rate of 500 pN/s. The results indicated stronger interaction between hydrophobins and hydrophobic surfaces than between two assembling hydrophobin molecules. Furthermore, this interaction was stable under different environmental conditions, which demonstrates the dominance of hydrophobicity in hydrophobin-hydrophobin interactions. This is the first time that interaction forces between hydrophobin molecules, and also between naturally occurring hydrophobic surfaces, have been measured directly at a single-molecule level.

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Linkers in polyproteins are considered as mere spacers between two adjacent domains. However, a series of studies using single-molecule force spectroscopy have recently reported distinct thermodynamic stability of I27 in polyproteins with varying linkers and indicated the vital role of linkers in domain stability. A flexible glycine rich linker (-(GGG)n, n ≥ 3) featured unfolding at lower forces than the regularly used arg-ser (RS) based linker. Interdomain interactions among I27 domains in Gly-rich linkers were suggested to lead to reduced domain stability. However, the negative impact of inter domain interactions on domain stability is thermodynamically counter-intuitive and demanded thorough investigations. Here, using an array of ensemble equilibrium experiments and in-silico measurements with I27 singlet and doublets with two aforementioned linkers, we delineate that the inter-domain interactions in fact raise the stability of the polyprotein with RS linker. More surprisingly, a highly flexible Gly-rich linker has no interference on the stability of polyprotein. Overall, we conclude that flexible linkers are preferred in a polyprotein for maintaining domain's independence.

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Complex physical cues including two-dimensional membrane environment, dynamic mechanical force, and bioelectric activity inevitably affect membrane receptor functions. Multiplexed single-molecule force spectroscopy (SMFS) techniques with the capability of live-cell measurements are essential to systemically dissect receptor's functions under complex biophysical regulation. In this review, we summarize recent progress of live-cell based SMFS techniques and specifically focus on the progress of SMFS on the biomembrane force probe with enhanced mechanical stability and multiplexed capability of fluorescence imaging. We further suggest the necessity of developing multiplexed SMFS techniques with simultaneous bioelectric regulation capability to investigate membrane potential regulated membrane receptor functions. These state-of-art multiplexed SMFS techniques will dissect membrane receptors functions in a systematic biophysical angle, resolving the biochemical, biomechanical and bioelectrical regulatory mechanisms in physiologically relevant conditions.

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Rhodopsin-like G protein-coupled receptors (GPCRs), widely distributed in microorganisms, invertebrates, and vertebrates, are the largest class in GPCRs, and are involved in many important physiological and pathological processes, including the photosensitivity, regulation of behavior and emotion, and so on. Atomic force microscopy (AFM) is a powerful and multifunctional toolkit in bionanotechnology, as it can image the morphology of membrane proteins at subnanometer spatial resolution and detect forces related with membrane proteins down to piconewton level by single-molecule force spectroscopy (SMFS) mode under physiological conditions. Herein, the achievements of AFM in the study of rhodopsin-like GPCRs, including observing the high-resolution topography and structural changes, revealing the interaction forces, binding kinetics, and mechanical properties (such as modulus), are reviewed and summarized. Finally, the challenges, outlook, and prospects of AFM in the study of rhodopsin-like GPCRs are discussed.

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Many proteins and peptides have been identified to effectively and specifically bind on certain surfaces such as silica, polystyrene and titanium dioxide. It is of great interest, in many areas such as enzyme immobilization, surface functionalization and nanotechnology, to understand how these proteins/peptides bind to solid surfaces. Here we use single-molecule force spectroscopy (SMFS) based on atomic force microscopy to directly measure the adhesion force between a silica-binding peptide SB7 and glass surface at single molecule level. SMFS results show that the adhesion force of a single SB7 detaching from the glass surface distributes in two populations at ~220 pN and 610 pN, which is higher than the unfolding forces of most mechanically stable proteins and the unbinding forces of most stable protein-protein interactions. Molecular dynamics simulation reveals that the electrostatic interactions between positively charged arginine residues and the silica surface dominates the binding of SB7 on silica. Our study provides experimental evidence and molecular mechanism at the single-molecule level for the SB7-based immobilization of proteins on silica-based surface, which is able to withstand high mechanical forces, making it an ideal fusion tag for silica surface immobilization or peptide-base adhesive materials.

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The worldwide emergence of antimicrobial resistance (AMR) in pathogenic microorganisms, including bacteria and viruses due to a plethora of reasons, such as genetic mutation and indiscriminate use of antimicrobials, is a major challenge faced by the healthcare sector today. One of the issues at hand is to effectively screen and isolate resistant strains from sensitive ones. Utilizing the distinct nanomechanical properties (e.g., elasticity, intracellular turgor pressure, and Young's modulus) of microbes can be an intriguing way to achieve this; while atomic force microscopy (AFM), with or without modification of the tips, presents an effective way to investigate such biophysical properties of microbial surfaces or an entire microbial cell. Additionally, advanced AFM instruments, apart from being compatible with aqueous environments-as often is the case for biological samples-can measure the adhesive forces acting between AFM tips/cantilevers (conjugated to bacterium/virion, substrates, and molecules) and target cells/surfaces to develop informative force-distance curves. Moreover, such force spectroscopies provide an idea of the nature of intercellular interactions (e.g., receptor-ligand) or propensity of microbes to aggregate into densely packed layers, that is, the formation of biofilms-a property of resistant strains (e.g., Staphylococcus aureus, Pseudomonas aeruginosa). This mini-review will revisit the use of single-cell force spectroscopy (SCFS) and single-molecule force spectroscopy (SMFS) that are emerging as powerful additions to the arsenal of researchers in the struggle against resistant microbes, identify their strengths and weakness and, finally, prioritize some future directions for research.

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Cadherin-23, a giant atypical cadherin, forms homophilic interactions at the cell-cell junction of epithelial cells and heterophilic interactions with protocadherin-15 at the tip-links of neuroepithelial cells. While the molecular structure of the heterodimer is solved, the homodimer structure is yet to be resolved. The homodimers play an essential role in cell-cell adhesion as the downregulation of cadherin-23 in cancers loosen the intercellular junction resulting in faster-migration of cancer cells and a significant drop in patient survival. In vitro studies have measured a stronger aggregation-propensity of cadherin-23 compared to typical E-cadherin. Here, we deciphered the unique trans-homodimer structure of cadherin-23 in solution, and show that it consists of two electrostatic-based interfaces extended up to two terminal domains. The interface is robust, with a low off-rate of ~8x10-4 s-1 that supports its strong aggregation-propensity. We identified a point-mutation, E78K, that disrupts this binding. Interestingly, a mutation at the interface was reported in skin cancer. Overall, the structural basis of the strong cadherin-23 adhesion may have far-reaching applications in the fields of mechanobiology and cancer.

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G protein-coupled receptors (GPCRs) show complex relationships between functional states and conformational plasticity that can be qualitatively and quantitatively described by contouring their free energy landscape. However, how ligands modulate the free energy landscape to direct conformation and function of GPCRs is not entirely understood. Here, we employ single-molecule force spectroscopy to parametrize the free energy landscape of the human protease-activated receptor 1 (PAR1), and delineate the mechanical, kinetic, and energetic properties of PAR1 being set into different functional states. Whereas in the inactive unliganded state PAR1 adopts mechanically rigid and stiff conformations, upon agonist or antagonist binding the receptor mechanically softens, while increasing its conformational flexibility, and kinetic and energetic stability. By mapping the free energy landscape to the PAR1 structure, we observe key structural regions putting this conformational plasticity into effect. Our insight, complemented with previously acquired knowledge on other GPCRs, outlines a more general framework to understand how GPCRs stabilize certain functional states.

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The application of equipment and tools that produce a magnetic field is increasing in aquatic ecosystems. In the present study, the effects of acute (1 week) and subacute (3 weeks) exposures to different static magnetic fields (SMFs) of 2.5, 5, 7.5 mT on stress indices (cortisol and glucose), sex steroid hormones (17ß-estradiol and 17-α hydroxy progesterone) and fecundity of the zebrafish (Danio rerio) were investigated. The obtained results showed a significant change in cortisol, glucose, 17ß-estradiol (E2) and 17-α hydroxy progesterone (17-OHP) levels by enhancing the intensity and time of exposure to SMFs. In conclusion, the SMFs, especially at higher levels of intensities, showed physiologically harmful effects on the reproductive biology of the zebrafish during acute and subacute exposures.

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Adenosine triphosphate (ATP), an indispensable molecule that provides energy for essentially all cellular processes, has been shown to be affected by some magnetic fields (MFs). Although people are frequently exposed to various static and power frequency MFs in their daily lives, the exact effects of these MFs of different frequencies have not been systematically investigated. Here, we tested 6-mT MFs with 0, 50, and 120 Hz for their effects on cellular ATP levels in 11 different cell lines. We found that the 6-mT static magnetic field (SMF) either does not affect or increase cellular ATP levels, while 6-mT 50-Hz MF either does not affect or decrease cellular ATP levels. In contrast, 6-mT 120-Hz MF has variable effects. We examined the mitochondrial membrane potential (MMP) as well as reactive oxygen species (ROS) in four different cell lines, but did not find their direct correlation with ATP levels. Although none of the ATP level changes induced by these three different frequencies of 6-mT MFs are dramatic, these results may be used to explain some differential cellular responses of various cell lines to different frequency MFs.

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The protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR) involved in hemostasis, thrombosis, and inflammation, is activated by thrombin or other coagulation proteases. This activation is inhibited by the irreversible antagonist vorapaxar used for anti-platelet therapy. Despite detailed structural and functional information, how vorapaxar binding alters the structural properties of PAR1 to prevent activation is hardly known. Here we apply dynamic single-molecule force spectroscopy to characterize how vorapaxar binding changes the mechanical, kinetic, and energetic properties of human PAR1 under physiologically relevant conditions. We detect structural segments stabilizing PAR1 and quantify their properties in the unliganded and the vorapaxar-bound state. In the presence of vorapaxar, most structural segments increase conformational variability, lifetime, and free energy, and reduce mechanical rigidity. These changes highlight a general trend in how GPCRs are affected by strong antagonists.

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