AFM-based Single-molecule Force Spectroscopy Research Articles

Single-Molecule-Level Quantification Based on Atomic Force Microscopy Data Reveals the Interaction between Melittin and Lipopolysaccharide in Gram-Negative Bacteria.

The interaction forces and mechanical properties of the interaction between melittin (Mel) and lipopolysaccharide (LPS) are considered to be crucial driving forces for Mel when killing Gram-negative bacteria (GNB). However, how their interaction forces perform at the single-molecule level and the dissociation kinetic characteristics of the Mel/LPS complex remain poorly understood. In this study, the single-molecule-level interaction forces between Mel and LPSs from E. coli K-12, O55:B5, O111:B4, and O128:B12 were explored using atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS). AFM-based dynamic force spectroscopy (DFS) and an advanced analytical model were employed to investigate the kinetic characteristics of the Mel/LPS complex dissociation. The results indicated that Mel could interact with both rough (R)-form LPS (E. coli K-12) and smooth (S)-form LPSs (E. coli O55:B5, O111:B4, and O128:B12). The S-form LPS showed a more robust interaction with Mel than the R-form LPS, and a slight difference existed in the interaction forces between Mel and the diverse S-form LPS. Mel interactions with the S-form LPSs showed greater specific and non-specific interaction forces than the R-form LPS (p < 0.05), as determined by AFM-based SMFS. However, there was no significant difference in the specific and non-specific interaction forces among the three samples of S-form LPSs (p > 0.05), indicating that the variability in the O-antigen did not affect the interaction between Mel and LPSs. The DFS result showed that the Mel/S-form LPS complexes had a lower dissociation rate constant, a shorter energy barrier width, a longer bond lifetime, and a higher energy barrier height, demonstrating that Mel interacted with S-form LPS to form more stable complexes. This research enhances the existing knowledge of the interaction micromechanics and kinetic characteristics of Mel and LPS at the single-molecule level. Our research may help with the design and evaluation of new anti-GNB drugs.

Molecular Crosstalk Between Adherens Junction Proteins, E-cadherin and Nectin-4

Cell-cell junctions formed by the association of cell adhesion molecules facilitate physiological events necessary for growth and development of multicellular organisms. Among them, cadherins and nectins organize and assemble to form adherens junction, which thereby mechanically couples interacting cells. A detailed understanding of the crosstalk involving these cell adhesion molecules is fundamental to the study of the various developmental processes. Although, cadherins and nectins can recruit each other in the adherens junction through an interplay of cytoplasmic adaptor molecules, here, we report a direct interaction between N-terminal extracellular domains of E-cadherin and nectin-4 as demonstrated by surface plasmon resonance (SPR) and Atomic Force Microscopy (AFM)-based single molecule force spectroscopy (SMFS). Kinetic studies using SPR demonstrate the binding between the ectodomains of E-cadherin and nectin-4 with a KD of 3.7 ± 0.7 µM and KD of 5.4 ± 0.2 µM (reciprocal experiment). AFM-based SMFS experiments also support interaction between the ectodomains of E-cadherin and nectin-4 with the koff value of 31.48 ± 1.53 s−1 and the lifetime of the complex of 0.036 ± 0.0026 s. We thus propose a cell adhesion mechanism mediated by E-cadherin and nectin-4, which can have functional significance in early embryogenesis as evident from the expression pattern of both the proteins during early development.

Exploring the impact of PEGylation on the cell-nanomicelle interactions by AFM-based single-molecule force spectroscopy and force tracing.

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.

Biosensing, Characterization of Biosensors, and Improved Drug Delivery Approaches Using Atomic Force Microscopy: A Review

Since its invention, atomic force microscopy (AFM) has come forth as a powerful member of the “scanning probe microscopy” (SPM) family and an unparallel platform for high-resolution imaging and characterization for inorganic and organic samples, especially biomolecules, biosensors, proteins, DNA, and live cells. AFM characterizes any sample by measuring interaction force between the AFM cantilever tip (the probe) and the sample surface, and it is advantageous over other SPM and electron micron microscopy techniques as it can visualize and characterize samples in liquid, ambient air, and vacuum. Therefore, it permits visualization of three-dimensional surface profiles of biological specimens in the near-physiological environment without sacrificing their native structures and functions and without using laborious sample preparation protocols such as freeze-drying, staining, metal coating, staining, or labeling. Biosensors are devices comprising a biological or biologically extracted material (assimilated in a physicochemical transducer) that are utilized to yield electronic signal proportional to the specific analyte concentration. These devices utilize particular biochemical reactions moderated by isolated tissues, enzymes, organelles, and immune system for detecting chemical compounds via thermal, optical, or electrical signals. Other than performing high-resolution imaging and nanomechanical characterization (e.g., determining Young’s modulus, adhesion, and deformation) of biosensors, AFM cantilever (with a ligand functionalized tip) can be transformed into a biosensor (microcantilever-based biosensors) to probe interactions with a particular receptors of choice on live cells at a single-molecule level (using AFM-based single-molecule force spectroscopy techniques) and determine interaction forces and binding kinetics of ligand receptor interactions. Targeted drug delivery systems or vehicles composed of nanoparticles are crucial in novel therapeutics. These systems leverage the idea of targeted delivery of the drug to the desired locations to reduce side effects. AFM is becoming an extremely useful tool in figuring out the topographical and nanomechanical properties of these nanoparticles and other drug delivery carriers. AFM also helps determine binding probabilities and interaction forces of these drug delivery carriers with the targeted receptors and choose the better agent for drug delivery vehicle by introducing competitive binding. In this review, we summarize contributions made by us and other researchers so far that showcase AFM as biosensors, to characterize other sensors, to improve drug delivery approaches, and to discuss future possibilities.

Multivalent non-covalent interactions lead to strongest polymer adhesion.

Multivalent interactions play a leading role in biological processes such as the inhibition of inflammation or virus internalization. The multivalent interactions show enhanced strength and better selectivity compared to monovalent interactions, but they are much less understood due to their complexity. Here, we detect molecular interactions in the range of a few piconewtons to several nanonewtons and correlate them with the formation and subsequent breaking of one or several bonds and assign these bonds. This becomes possible by performing atomic force microcopy (AFM)-based single molecule force spectroscopy of a multifunctional polymer covalently attached to an AFM cantilever tip on a substrate bound polymer layer of the multifunctional polymer. Varying the pH value and the crosslinking state of the polymer layer, we find that bonds of intermediate strength (non-covalent), like coordination bonds, give the highest multivalent bond strength, even outperforming strong (covalent) bonds. At the same time, covalent bonds enhance the polymer layer density, increasing in particular the number of non-covalent bonds. In summary, we can show that the key for the design of stable and durable polymer coatings is to provide a variety of multivalent interactions and to keep the number of non-covalent interactions at a high level.

Exploring the Impact of PEGylation on the Cell-Nanomicelle Interactions by AFM-Based Single-Molecule Force Spectroscopy and Force Tracing

Exploring the Impact of PEGylation on the Cell-Nanomicelle Interactions by AFM-Based Single-Molecule Force Spectroscopy and Force Tracing

Facile Synthesis of Peptide-Conjugated Gold Nanoclusters with Different Lengths.

The synthesis of ultra-small gold nanoclusters (Au NCs) with sizes down to 2 nm has received increasing interest due to their unique optical and electronic properties. Like many peptide-coated gold nanospheres synthesized before, modified gold nanoclusters with peptide conjugation are potentially significant in biomedical and catalytic fields. Here, we explore whether such small-sized gold nanoclusters can be conjugated with peptides also and characterize them using atomic force microscopy. Using a long and flexible elastin-like polypeptide (ELP)20 as the conjugated peptide, (ELP)20-Au NCs was successfully synthesized via a one-pot synthesis method. The unique optical and electronic properties of gold nanoclusters are still preserved, while a much larger size was obtained as expected due to the peptide conjugation. In addition, a short and rigid peptide (EAAAK)3 was conjugated to the gold nanoclusters. Their Yong’s modulus was characterized using atomic force microscopy (AFM). Moreover, the coated peptide on the nanoclusters was pulled using AFM-based single molecule-force spectroscopy (SMFS), showing expected properties as one of the first force spectroscopy experiments on peptide-coated nanoclusters. Our results pave the way for further modification of nanoclusters based on the conjugated peptides and show a new method to characterize these materials using AFM-SMFS.

Revealing the Underestimated Anticancer Effect of Azurin by Mechanical Unfolding.

As a potential anticancer agent, azurin has attracted extensive attraction among chemists, physicists, and material scientists. Its structural and unfolding/folding information has been partially understood, but some detailed information, such as the difference in the unfolding processes between apo-azurin and holo-azurin, the mechanical stability, and the role of the copper cluster in its stability, has not been addressed adequately, especially at the single-molecule level. Here, we employed AFM-based single-molecule force spectroscopy to investigate the unfolding process of azurin in the apo and holo forms under an external force. The results indicated that the unfolding processes of apo-azurin and holo-azurin are different, and holo-azurin requires a stronger force to unfold than does apo-azurin. The copper cluster exhibited a more significant impact on the stability and the folding process of holo-azurin: the copper cluster was completely broken, and the copper ion left the unfolded azurin during the unfolding process of azurin. We suspected that the presence of the disulfide bond in azurin made the unfolding of the copper cluster different from that in pseudoazurin, which is also a type I copper protein like azurin. Rarely reported in previous studies, the mechanical strength of the Cu-N(His) bond of the copper cluster was obtained in this study, which is weaker than that of most metal-S(Cys) bonds but higher than that of the Fe-N(His) bond. Altogether, our results offer a possible new scenario for azurin to widely extend its anticancer activity.

Reconstruction of mechanical unfolding and refolding pathways of proteins with atomic force spectroscopy and computer simulations

Most proteins in proteomes are large, typically consist of more than one domain and are structurally complex. This often makes studying their mechanical unfolding pathways challenging. Proteins composed of tandem repeat domains are a subgroup of multi-domain proteins that, when stretched, display a saw-tooth pattern in their mechanical unfolding force extension profiles due to their repetitive structure. However, the assignment of force peaks to specific repeats undergoing mechanical unraveling is complicated because all repeats are similar and they interact with their neighbors and form a contiguous tertiary structure. Here, we describe in detail a combination of experimental and computational single-molecule force spectroscopy methods that proved useful for examining the mechanical unfolding and refolding pathways of ankyrin repeat proteins. Specifically, we explain and delineate the use of atomic force microscope-based single molecule force spectroscopy (SMFS) to record the mechanical unfolding behavior of ankyrin repeat proteins and capture their unusually strong refolding propensity that is responsible for generating impressive refolding force peaks. We also describe Coarse Grain Steered Molecular Dynamic (CG-SMD) simulations which complement the experimental observations and provide insights in understanding the unfolding and refolding of these proteins. In addition, we advocate the use of novel coiled-coils-based mechanical polypeptide probes which we developed to demonstrate the vectorial character of folding and refolding of these repeat proteins. The combination of AFM-based SMFS on native and CC-equipped proteins with CG-SMD simulations is powerful not only for ankyrin repeat polypeptides, but also for other repeat proteins and more generally to various multidomain, non-repetitive proteins with complex topologies.

Single-molecule mechanics of synthetic aromatic amide helices: Ultrafast and robust non-dissipative winding

Summary Because of proteins’ many degrees of conformational freedom, programming protein folding dynamics, overall elasticity, and motor functions remains an elusive objective. Instead, smaller and simpler objects, such as synthetic foldamers, may be amenable to design. However, little is known about their mechanical performance. Here, we show that reducing molecular size may not compromise mechanical properties. We report that helical aromatic oligoamides as small as 1 nm possess outstanding elasticity and outperform most natural helices. Using single-molecule force spectroscopy, we characterize their folding trajectories and intermediate states. We show that they cooperatively and reversibly unwind at high forces. They extend up to 3.8 times their original length and rewind against considerable forces on a timescale of 10 μs. Pulling and relaxing cycles follow the same trace up to a very high loading rate, indicating that the mechanical energy accumulated during the stretching does not dissipate and is immediately reusable.

Real-Time Fluctuations in Single-Molecule Rotaxane Experiments Reveal an Intermediate Weak Binding State during Shuttling.

We report on the use of atomic force microscopy (AFM) to identify and characterize an intermediate state in macrocycle shuttling in a hydrogen bonded amide-based molecular shuttle. The [2]rotaxane consists of a benzylic amide macrocycle mechanically locked onto a thread that bears both fumaramide and succinic amide-ester sites, each of which can bind to the macrocycle through up to four intercomponent hydrogen bonds. Using AFM-based single-molecule force spectroscopy, we mechanically triggered the translocation of the ring between the two principal binding sites ("stations") on the axle. Equilibrium fluctuations reveal another interacting site involving the two oxygen atoms in the middle of the thread. We characterized the ring occupancy distribution over time, which confirms the intermediate in both shuttling directions. The study provides evidence of weak hydrogen bonds that are difficult to detect using other methods and shows how the composition of the thread can significantly influence the shuttling dynamics by slowing down the ring motion between the principal binding sites. More generally, the study illustrates the utility that single-molecule experiments, such as force spectroscopy, can offer for elucidating the structure and dynamics of synthetic molecular machines.

Long-Range Ordered Water Correlations between A–T/C–G Nucleotides

Summary The interactions between complementary base pairs are crucial to the helical duplex structure of DNA. Although base-base interactions have been widely reported, the study of long-range interactions in the base-pairing processes is still a major challenge in this field. Here, we first study the long-range interactions between the base pairs by atomic force microscopy-based single-molecule force spectroscopy (SMFS). The SMFS results of A–T/C–G imply that there are weak long-range interactions between them, which have a range of 15–25 nm. Raman spectroscopy results imply that these weak interactions can be attributed to multiplex hydrogen bond interaction of ordered water structure between A–T/C–G nucleotides. In addition, the theoretical calculations deduce that the ideal structure of these water molecules exhibits a specific helical structure. Our findings might open up a new understanding of biological assembly processes and provide helpful strategies for bio-nanotechnology.

Blue light-induced low mechanical stability of ruthenium-based coordination bonds: an AFM-based single-molecule force spectroscopy study.

Metal complexes containing coordination bonds play a prominent role in many essential biological systems in living organisms. Examples of such complexes include hemoglobin containing iron, chlorophyll containing magnesium, and vitamin B12 containing cobalt. Although the thermodynamic and other collective properties of metal complexes are well established, their mechanical stability remains minimally explored. Single-molecule force spectroscopy has been used to determine the structural and mechanical properties of chemical bonds; however, it has been minimally utilized in the field of coordination chemistry. Thus, here, we select a unique molecule of interest, HA–RuII, {HA = hyaluronan and RuII = [(bpy)2Ru(4-pyNH2)2](PF6)2} and subject it to single-molecule force spectroscopy analysis to directly study its bond-rupture process. The molecule is excited by blue-light irradiation, and surprisingly, this whole process could be reversed without applying any external energy, such as heat or solvent exposure. Our results demonstrate the reversibility of the luminescent RuII complex to its original state, a phenomenon that can be further applied to other coordination compounds.

Multistep Protein Unfolding Scenarios from the Rupture of a Complex Metal Cluster Cd3S9

Protein (un)folding is a complex and essential process. With the rapid development of single-molecule techniques, we can detect multiple and transient proteins (un)folding pathways/intermediates. However, the observation of multiple multistep (>2) unfolding scenarios for a single protein domain remains limited. Here, we chose metalloprotein with relatively stable and multiple metal-ligand coordination bonds as a system for such a purpose. Using AFM-based single-molecule force spectroscopy (SMFS), we successfully demonstrated the complex and multistep protein unfolding scenarios of the β-domain of a human protein metallothionein-3 (MT). MT is a protein of ~60 amino acids (aa) in length with 20 cysteines for various metal binding, and the β-domain (βMT) is of ~30 aa with an M3S9 metal cluster. We detected four different types of three-step protein unfolding scenarios from the Cd-βMT, which can be possibly explained by the rupture of Cd-S bonds in the complex Cd3S9 metal cluster. In addition, complex unfolding scenarios with four rupture peaks were observed. The Cd-S bonds ruptured in both single bond and multiple bonds modes. Our results provide not only evidence for multistep protein unfolding phenomena but also reveal unique properties of metalloprotein system using single-molecule AFM.

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. Atomic force microscopy (AFM) can address this problem by enabling stretching and relaxation of single protein molecules, which gives direct evidence of specific unfolding and refolding characteristics. AFM-based single-molecule force-spectroscopy (AFM-SMFS) provides a means to consistently measure high-energy conformations in proteins that are not possible in traditional bulk (biochemical) measurements. Although numerous papers were published to show principles of AFM-SMFS, it is not easy to conduct SMFS experiments due to a lack of an exhaustively complete protocol. In this study, we briefly illustrate the principles of AFM and extensively detail the protocols, procedures, and data analysis as a guideline to achieve good results from SMFS experiments. We demonstrate representative SMFS results of single protein mechanical unfolding measurements and we provide troubleshooting strategies for some commonly encountered problems.

Single-molecule force spectroscopy to decipher the early signalling step in membrane-bound penicillin receptors embedded into a lipid bilayer.

Understanding the molecular mechanism by which the signal of the presence of an antibiotic is transduced from outside to inside the bacterial cell is of fundamental interest for the β-lactam antibiotic resistance problem, but remains difficult to accomplish. No approach has ever addressed entire penicillin receptors in a membrane environment. Here we describe a method to investigate the purified Bacillus licheniformis BlaR1 receptor -a membrane-bound penicillin receptor involved in β-lactam resistance- embedded into a lipid bilayer in absence or presence of penicillin. By selecting a mutated receptor blocked in its signal transduction pathway just after its activation by penicillin, we revealed the very first step of receptor signalling by unfolding the receptor from its C-terminal end by AFM-based single-molecule force spectroscopy. We showed that the presence of the antibiotic entails significant conformational changes within the receptor. Our approach opens an avenue to study signal-transduction pathways mediated by membrane-bound proteins in a membrane environment.

High-Precision Single-Molecule Characterization of the Folding of an HIV RNA Hairpin by Atomic Force Microscopy.

The folding of RNA into a wide range of structures is essential for its diverse biological functions from enzymatic catalysis to ligand binding and gene regulation. The unfolding and refolding of individual RNA molecules can be probed by single-molecule force spectroscopy (SMFS), enabling detailed characterization of the conformational dynamics of the molecule as well as the free-energy landscape underlying folding. Historically, high-precision SMFS studies of RNA have been limited to custom-built optical traps. Although commercial atomic force microscopes (AFMs) are widely deployed and offer significant advantages in ease-of-use over custom-built optical traps, traditional AFM-based SMFS lacks the sensitivity and stability to characterize individual RNA molecules precisely. Here, we developed a high-precision SMFS assay to study RNA folding using a commercial AFM and applied it to characterize a small RNA hairpin from HIV that plays a key role in stimulating programmed ribosomal frameshifting. We achieved rapid data acquisition in a dynamic assay, unfolding and then refolding the same individual hairpin more than 1,100 times in 15 min. In comparison to measurements using optical traps, our AFM-based assay featured a stiffer force probe and a less compliant construct, providing a complementary measurement regime that dramatically accelerated equilibrium folding dynamics. Not only did kinetic analysis of equilibrium trajectories of the HIV RNA hairpin yield the traditional parameters used to characterize folding by SMFS (zero-force rate constants and distances to the transition state), but we also reconstructed the full 1D projection of the folding free-energy landscape comparable to state-of-the-art studies using dual-beam optical traps, a first for this RNA hairpin and AFM studies of nucleic acids in general. Looking forward, we anticipate that the ease-of-use of our high-precision assay implemented on a commercial AFM will accelerate studying folding of diverse nucleic acid structures.

Detecting van der Waals forces between a single polymer repeating unit and a solid surface in high vacuum

Ubiquitous van der Waals (vdW) forces are very important for nanostructures. Although the vdW forces between two surfaces (or two layers) have been measured for several decades, a direct detection at the single-molecule level is still difficult. Herein, we report a novel method to solve this problem in high vacuum by means of AFM-based single-molecule force spectroscopy (SMFS). Solvent molecules and surface adsorbed water are removed thoroughly under high vacuum so that the situation is greatly simplified. A constant force plateau can be observed when a polymer chain is peeled off from a substrate in high vacuum. Accordingly, the vdW forces between one polymer repeating unit and the substrates can be obtained. The experimental results show that the vdW forces (typical range: 21–54 pN) are dependent on the species of substrates and the size of polymer repeating unit, which is in good accordance with the theoretical results. It is expected that this novel method can be applied to detect other non-covalent interactions (such as hydrogen bond and π-π stacking) at the single-molecule level in the future.

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

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.

Single-Molecule Force Spectroscopy Reveals Self-Assembly Enhanced Surface Binding of Hydrophobins.

Hydrophobins have raised lots of interest as powerful surface adhesives. However, it remains largely unexplored how their strong and versatile surface adhesion is linked to their unique amphiphilic structural features. Here, we develop an AFM-based single-molecule force spectroscopy assay to quantitatively measure the binding strength of hydrophobin to various types of surfaces both in isolation and in preformed protein films. We find that individual class II hydrophobins (HFBI) bind strongly to hydrophobic surfaces but weakly to hydrophilic ones. After self-assembly into protein films, they show much stronger binding strength to both surfaces due to the cooperativity of different interactions at nanoscale. Such self-assembly enhanced surface binding may serve as a general design principle for synthetic bioactive adhesives.