Because of its piconewton force sensitivity and nanometer positional accuracy, the atomic force microscope (AFM) has emerged as a powerful tool for exploring the forces and the dynamics of the ...interaction between individual ligands and receptors, either on isolated molecules or on cellular surfaces. These studies require attaching specific biomolecules or cells on AFM tips and on solid supports and measuring the unbinding forces between the modified surfaces using AFM force spectroscopy. In this review, we describe the current methodology for molecular recognition studies using the AFM, with an emphasis on strategies available for preparing AFM tips and samples, and on procedures for detecting and localizing single molecular recognition events.
Elucidation the nano-organization of membrane proteins at/within the plasma membrane is probably the most demanding and still challenging task in cell biology since requires experimental approaches ...with nanoscale resolution. During last decade, atomic force microscopy (AFM)-based simultaneous topography and recognition imaging (TREC) has become a powerful tool to quickly obtain local receptor nano-maps on complex heterogeneous biosurfaces such as cells and membranes. Here we emphasize the TREC technique and explain how to unravel the nano-landscape of mammalian cells. We describe the procedures for all steps of the experiment including tip functionalization with ligand molecules, sample preparation, and localization of key molecules on the cell surface. We also discuss the current limitations and future perspectives of this technique.
Microbial resistant coatings have raised considerable interest in the biotechnological industry and clinical scenarios to combat the spreading of infections, in particular in implanted medical ...devices. Polymer brushes covalently attached to surfaces represent a useful platform to identify ideal compositions for preventing bacterial settlement by quantifying bacteria–surface interactions. In this work, a series of polymer brushes with different charges, positively charged poly2-(methacryloyloxy)ethyl trimethylammonium chloride (PMETAC), negatively charged poly(3-sulfopropyl methacrylate potassium salt) (PSPMA), and neutral poly(2-hydroxyethyl methacrylate) (PHEMA) were grafted onto glass surfaces by surface-initiated atom transfer radical polymerization in aqueous conditions. The antimicrobial activity of the polymer brushes against Gram-negative Escherichia coli was tested at the nano- and microscopic level on different time scales, that is, from nm to 100 μm, and ms to 24 h, respectively. The interaction between the polymer brushes and E. coli was studied by single-cell force spectroscopy (SCFS) and by quantification of the bacterial density on surfaces incubated with bacterial suspensions. E. coli firmly attached to positive PMETAC brushes with high work required for de-adhesion of 28 ± 9 nN·nm, but did not significantly bind to negatively charged PSPMA and neutral PHEMA brushes. Our studies of bacterial adhesion using polymer brushes with controllable chemistry provide essential insights into bacterial surface interactions and the origins of bacterial adhesion.
IgG antibodies play a central role in protection against pathogens by their ability to alert and activate the innate immune system. Here, we show that IgGs assemble into oligomers on antigenic ...surfaces through an ordered, Fc domain-mediated process that can be modulated by protein engineering. Using high-speed atomic force microscopy, we unraveled the molecular events of IgG oligomer formation on surfaces. IgG molecules were recruited from solution although assembly of monovalently binding molecules also occurred through lateral diffusion. Monomers were observed to assemble into hexamers with all intermediates detected, but in which only hexamers bound C1. Functional characterization of oligomers on cells also demonstrated that C1 binding to IgG hexamers was a prerequisite for maximal activation, whereas tetramers, trimers, and dimers were mostly inactive. We present a dynamic IgG oligomerization model, which provides a framework for exploiting the macromolecular assembly of IgGs on surfaces for tool, immunotherapy, and vaccine design.
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.
The flexibilities of extracellular loops determine ligand binding and activation of membrane receptors. Arising from fluctuations in inter- and intraproteinaceous interactions, flexibility manifests ...in thermal motion. Here we demonstrate that quantitative flexibility values can be extracted from directly imaging the thermal motion of membrane protein moieties using high-speed atomic force microscopy (HS-AFM). Stiffness maps of the main periplasmic loops of single reconstituted water channels (AqpZ, GlpF) revealed the spatial and temporal organization of loop-stabilizing intraproteinaceous H-bonds and salt bridges.
Recent waves of COVID-19 correlate with the emergence of the Delta and the Omicron variant. We report that the Spike trimer acts as a highly dynamic molecular caliper, thereby forming up to three ...tight bonds through its RBDs with ACE2 expressed on the cell surface. The Spike of both Delta and Omicron (B.1.1.529) Variant enhance and markedly prolong viral attachment to the host cell receptor ACE2, as opposed to the early Wuhan-1 isolate. Delta Spike shows rapid binding of all three Spike RBDs to three different ACE2 molecules with considerably increased bond lifetime when compared to the reference strain, thereby significantly amplifying avidity. Intriguingly, Omicron (B.1.1.529) Spike displays less multivalent bindings to ACE2 molecules, yet with a ten time longer bond lifetime than Delta. Delta and Omicron (B.1.1.529) Spike variants enhance and prolong viral attachment to the host, which likely not only increases the rate of viral uptake, but also enhances the resistance of the variants against host-cell detachment by shear forces such as airflow, mucus or blood flow. We uncover distinct binding mechanisms and strategies at single-molecule resolution, employed by circulating SARS-CoV-2 variants to enhance infectivity and viral transmission.
Influenza virus belongs to a wide range of enveloped viruses. The major spike protein hemagglutinin binds sialic acid residues of glycoproteins and glycolipids with dissociation constants in the ...millimolar range Sauter NK, et al. (1992) Biochemistry 31:9609–9621, indicating a multivalent binding mode. Here, we characterized the attachment of influenza virus to host cell receptors using three independent approaches. Optical tweezers and atomic force microscopy-based single-molecule force spectroscopy revealed very low interaction forces. Further, the observation of sequential unbinding events strongly suggests a multivalent binding mode between virus and cell membrane. Molecular dynamics simulations reveal a variety of unbinding pathways that indicate a highly dynamic interaction between HA and its receptor, allowing rationalization of influenza virus–cell binding quantitatively at the molecular level.
Activation of membrane receptors through clustering is a common mechanism found in various biological systems. Spatial proximity of receptors may be transduced across the membrane to initiate ...signaling pathways or alternatively be recognized by peripheral proteins or immune cells to trigger external effector functions. Here we show how specific immunoglobulin G (IgG) binding induces clustering of monomeric target molecules in lipid membranes through Fc–Fc interactions. We visualize and characterize the dynamic IgG oligomerization process and the molecular interactions involved using high-speed atomic force microscopy, single-molecule force spectroscopy, and quartz crystal microbalance experiments. We found that the Fc–Fc interaction strength is precisely tuned to be weak enough to prevent IgG oligomerization in solution at physiological titers, but enabling IgG oligomerization when Fabs additionally bind to their cognate surface epitopes, a mechanism that ultimately targets IgG-mediated effector functions such as classical complement activation to antigenic membranes.
Lipopolysaccharide (LPS) on gram-negative bacterial outer membranes is the first target for antimicrobial agents, due to their spatial proximity to outer environments of microorganisms. To develop ...antibacterial compounds with high specificity for LPS binding, the understanding of the molecular nature and their mode of recognition is of key importance. In this study, atomic force microscopy (AFM) and single molecular force spectroscopy were used to characterize the effects of antibiotic polymyxin B (PMB) to the bacterial membrane at the nanoscale. Isolated LPS layer and the intact bacterial membrane were examined with respect to morphological changes at different concentrations of PMB. Our results revealed that 3 hours of 10 μg/mL of PMB exposure caused the highest roughness changes on intact bacterial surfaces, arising from the direct binding of PMB to LPS on the bacterial membrane. Single molecular force spectroscopy was used to probe specific interaction forces between the isolated LPS layer and PMB coupled to the AFM tip. A short range interaction regime mediated by electrostatic forces was visible. Unbinding forces between isolated LPS and PMB were about 30 pN at a retraction velocity of 500 nm/s. We further investigated the effects of the polycationic peptide PMB on bacterial outer membranes and monitored its influences on the deterioration of the bacterial membrane structure. Polymyxin B binding led to rougher appearances and wrinkles on the outer membranes surface, which may finally lead to lethal membrane damage of bacteria. Our studies indicate the potential of AFM for applications in pathogen recognition and nano-resolution approaches in microbiology.
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