Single-molecule Dynamic Force Spectroscopy Research Articles

Exploring how lignin structure influences the interaction between carbohydrate-binding module and lignin using AFM

Nonproductive adsorption of cellulase onto the residual lignin in substrate seriously hinders the enzymatic hydrolysis. To understand how lignin structure affects lignin-cellulase interaction, the carbohydrate-binding module (CBM) functionalized atomic force microscope tip was used to measure CBM-lignin interaction by single-molecule dynamic force spectroscopy in this work. The results showed that sulfonated lignin (SL) has the greatest adhesion force to CBM (4.74 nN), while those of masson pine milled wood lignin (MWL), poplar MWL and herbaceous MWLs were 2.85, 1.03 and 0.27–0.61 nN, respectively. It provides direct quantitative evidence for the significance of lignin structure on lignin-cellulase interaction. The CBM-MWLs interaction decreased sharply to 0.054–0.083 nN while SL was added, indicating the primary mechanism of SL promoting lignocellulose hydrolysis was significantly reducing the nonproductive adsorption of substrate lignin on cellulase. Finally, the “competitive adsorption” mechanism was proposed to interpret why SL effectively promotes the enzymatic hydrolysis of lignin-containing substrates.

Ligand-Receptor Binding on Cell Membrane: Dynamic Force Spectroscopy Applications.

Ligand-receptor recognition on the cell membrane enables the communication of cells with the extracellular environment. Atomic force microscopy (AFM)-based single-molecule dynamic force spectroscopy represents one of the most powerful techniques available to directly investigate ligand-receptor recognition under physiological conditions without considerable disruption to cells. It provides important information for research on biological processes, disease pathogenesis, and mechanism of drugs. Here we describe an example of applying single-molecule dynamic force spectroscopy to study the binding of epidermal growth factor (EGF) to its receptor EGFR, as well as the effect of two clinical drugs, Pertuzumab and Trastuzumab, on the interaction of EGF and EGFR.

Interactions of Histone Acetyltransferase p300 with the Nuclear Proteins Histone and HMGB1, As Revealed by Single Molecule Atomic Force Spectroscopy.

One of the important properties of the transcriptional coactivator p300 is histone acetyltransferase (HAT) activity that enables p300 to influence chromatin action via histone modulation. p300 can exert its HAT action upon the other nuclear proteins too--one notable example being the transcription-factor-like protein HMGB1, which functions also as a cytokine, and whose accumulation in the cytoplasm, as a response to tissue damage, is triggered by its acetylation. Hitherto, no information on the structure and stability of the complexes between full-length p300 (p300FL) (300 kDa) and the histone/HMGB1 proteins are available, probably due to the presence of unstructured regions within p300FL that makes it difficult to be crystallized. Herein, we have adopted the high-resolution atomic force microscopy (AFM) approach, which allows molecularly resolved three-dimensional contour mapping of a protein molecule of any size and structure. From the off-rate and activation barrier values, obtained using single molecule dynamic force spectroscopy, the biochemical proposition of preferential binding of p300FL to histone H3, compared to the octameric histone, can be validated. Importantly, from the energy landscape of the dissociation events, a model for the p300-histone and the p300-HMGB1 dynamic complexes that HAT forms, can be proposed. The lower unbinding forces of the complexes observed in acetylating conditions, compared to those observed in non-acetylating conditions, indicate that upon acetylation, p300 tends to weakly associate, probably as an outcome of charge alterations on the histone/HMGB1 surface and/or acetylation-induced conformational changes. To our knowledge, for the first time, a single molecule level treatment of the interactions of HAT, where the full-length protein is considered, is being reported.

Crucial Roles of Bond Rebinding in Rupture Behaviors of Single Molecular Bond at Ultralow Loading Rates

Single-molecule dynamic force spectroscopy (DFS) is a powerful tool for studying mechanical forces of molecular interactions. Recently, the important role of bond rebinding in DSF experiments was recognized, which intrigued mounting researches in this direction. In this work, we study how the bond rebinding influences the strength of single-molecular bonds using Brownian dynamics (BD) simulations and theoretical modeling. Our results show that bond rebinding significantly enhances the strength of single-molecular bond at ultralow loading rates. The rebinding behavior strongly depends on the loading stiffness, suggesting that the strength of single-molecular bond is not only dependent on its intrinsic property, but also the stiffness of loading device. By connecting our new model with conventional theories that did not consider the rebinding effect and are only applied to high loading rates, we propose a unified scheme to predict the rupture forces in a full range of loading rate in DSF experiments and simulations.

Surface conformations of an anti-ricin aptamer and its affinity for ricin determined by atomic force microscopy and surface plasmon resonance.

We used atomic force microscopy (AFM) and surface plasmon resonance (SPR) to study the surface conformations of an anti-ricin aptamer and its specific binding affinity for ricin molecules. The effect of surface modification of the Au(111) substrate on the aptamer affinity was also estimated. The AFM topography images had a resolution high enough to distinguish different aptamer conformations. The specific binding site on the aptamer molecule was clearly located by the AFM recognition images. The aptamer on a Au(111) surface modified with carboxymethylated-dextran (CD) showed both similarities to and differences from the one without CD modification. The influence of CD modification was evaluated using AFM images of various aptamer conformations on the Au(111) surface. The affinity between ricin and the anti-ricin aptamer was estimated using the off-rate values measured using AFM and SPR. The SPR measurements of the ricin sample were conducted in the range from 83.3 pM to 8.33 nM, and the limit of detection was estimated as 25 pM (1.5 ng mL(-1)). The off-rate values of the ricin-aptamer interactions were estimated using both single-molecule dynamic force spectroscopy (DFS) and SPR as (7.3 ± 0.4) × 10(-4) s(-1) and (1.82 ± 0.067) × 10(-2) s(-1), respectively. The results show that single-molecule measurements can obtain different reaction parameters from bulk solution measurements. In AFM single-molecule measurements, the various conformations of the aptamer immobilized on the gold surface determined the availability of each specific binding site to the ricin molecules. The SPR bulk solution measurements averaged the signals from specific and non-specific interactions. AFM images and DFS measurements provide more specific information on the interactions of individual aptamer and ricin molecules.

Probing Protein-Lipid Interactions at the Single Molecule Level

Cell membranes consist mostly of lipids and embedded proteins, forming a shell around the cell that controls information and material fluxes into and out of the cell. Protein-lipid interactions thus play a crucial role in numerous cellular mechanisms. Understanding these interactions would provide basic biophysical knowledge in addition to advance pharmaceutical developments, many of which target membranes. Despite their significance, protein-lipid interactions are not fully understood and have not yet been fully investigated from a mechanistic standpoint. In this study we investigated synthesized peptide constructs that insert into lipid bilayers via force spectroscopy. The constructs used here are based on SecA, a membrane-associated ATPase of the general secretory system. We investigated the first 10 amino terminal residues of SecA (SecA2-11) in three different arrangements to provide control experiments and guide interpretation. In one arrangement, two copies of SecA2-11 were covalently linked in series to the atomic force microscope tip, in another, they were linked in parallel. In all cases the constructs were brought into close proximity of a supported POPC lipid bilayer and the mechanical interaction between the peptide and lipid was recorded using single-molecule dynamic force spectroscopy. Investigating chemically similar yet geometrically different constructs provides important benchmarks for the interpretation of future studies involving longer constructs such as full length SecA.

Force-induced globule-coil transition in laminin binding protein and its role for viral-cell membrane fusion.

The specific interactions of the pairs laminin binding protein (LBP)-purified tick-borne encephalitis viral surface protein E and certain recombinant fragments of this protein, as well as West Nile viral surface protein E and certain recombinant fragments of that protein, are studied by combined methods of single-molecule dynamic force spectroscopy (SMDFS), enzyme immunoassay and optical surface waves-based biosensor measurements. The experiments were performed at neutral pH (7.4) and acid pH (5.3) conditions. The data obtained confirm the role of LBP as a cell receptor for two typical viral species of the Flavivirus genus. A comparison of these data with similar data obtained for another cell receptor of this family, namely human αVβ3 integrin, reveals that both these receptors are very important. Studying the specific interaction between the cell receptors in question and specially prepared monoclonal antibodies against them, we could show that both interaction sites involved in the process of virus-cell interaction remain intact at pH 5.3. At the same time, for these acid conditions characteristic for an endosome during flavivirus-cell membrane fusion, SMDFS data reveal the existence of a force-induced (effective already for forces as small as 30-70 pN) sharp globule-coil transition for LBP and LBP-fragments of protein E complexes. We argue that this conformational transformation, being an analog of abrupt first-order phase transition and having similarity with the famous Rayleigh hydrodynamic instability, might be indispensable for the flavivirus-cell membrane fusion process.

Comparison of three competing dynamic force spectroscopy models to study binding forces of amyloid-β (1–42)

We performed single molecule dynamic force spectroscopy experiments to study the dimerization of two amyloid-β (1-42) peptides and compared three different theoretical models used to fit experimental data: Bell-Evans, Dudko-Hummer-Szabo, and Friddle-De Yoreo. Using these models we extracted values of the dissociation rate at zero force, k0, and height and the width of the energy barrier, ΔG and xβ. We show the importance of including the effect of the linker molecule. All three models corrected for the linker effect give comparable results for xβ and show more discrepancy for k0 and ΔG values, ΔG parameter correlates well between Dudko-Hummer-Szabo and Friddle-De Yoreo models but differs for the Bell-Evans model.

Dependence of the most probable and average bond rupture force on the force loading rate: First order correction to the Bell–Evans model

The Bell–Evans model which predicts the linear dependence of the most probable intermolecular bond rupture force on the logarithm of stretching force loading rate is usually used to discuss the dynamic force spectroscopy experiment data. This model is consistent with the Kramers' theory of the bond dissociation rate only if one presupposes an independence of the pre-exponential factors in the Kramers relation on the acting force and a linear decrease of the dissociation barrier height on this same force, and for this to be true rather special shape of the interaction landscape is required. Here, we present a first order correction to this model (first terms of corresponding Taylor expansions are taken into account), discuss its implication for the interpretation of dynamic force spectroscopy experiment data and compare our model with the Monte Carlo simulation of a specially designed single molecule dynamic force spectroscopy experiment. In addition to the most probable bond rupture force, an average rupture force values are also calculated. All approximations made and the range of applicability of the obtained results are carefully described and compared with those for some other models in the field.

Measurements of single molecular affinity interactions between carbohydrate-binding modules and crystalline cellulose fibrils

Combining atomic force microscopy (AFM) recognition imaging and single molecule dynamic force spectroscopy (SMDFS), we studied the single molecule affinity interactions between the carbohydrate-binding module (CBM) and plant cell wall cellulose using the CBM3a (from Clostridium thermocellum) and CBM2a (from Cellvibrio japonicus) functionalized AFM tips. The binding efficiencies of the CBMs to the cellulose were determined by the binding areas on the crystalline cellulose fibrils surface using the recognition imaging. Several dynamic and kinetic parameters, such as the reconstructed free energy change, energy barrier and bond lifetime constant, were also obtained based on the measured single molecule unbinding forces, which are used to illuminate the affinity of the CBMs binding to the natural and single cellulose surface from a totally different aspect. It was found that CBM3a has a little higher binding efficiency and affinity than CBM2a to both natural and extracted cellulose surfaces and both the CBMs have higher affinities to the natural cell wall cellulose compared to the extracted single cellulose. The in-depth understanding of the binding mechanisms of the CBM-cellulose interactions of this study may pave the way for more efficient plant cell wall degradation and eventually facilitate biofuel production.

Dependence of the Most Probable and Average Bond Rupture Forces on the Force Loading Rate in Single Molecule Dynamic Force Spectroscopy Experiment: First Order Correction to the Bell - Evans Model

The Bell-Evans model which predicts the linear dependence of the most probable bond rupture force on the logarithm of the force loading rate is usually used to discuss the dynamic force spectroscopy experiment data. This model is consistent with the Kramers' theory of the bond dissociation rate only if one presupposes an independence of the pre-exponential factors in the Kramers relation on the acting force F and a linear decrease of the dissociation barrier height on this same force, and for this to be true a rather special shape of the interaction landscape is required.

Imaging and Measuring Single-Molecule Interaction between a Carbohydrate-Binding Module and Natural Plant Cell Wall Cellulose

The affinitive interaction between a carbohydrate-binding module (CBM3a) and natural crystalline cellulose was visualized and measured at the single-molecule level. Noncontact high resolution imaging by atomic force microscopy (AFM) was used to follow the binding process, in real time, of CBM3a-functionalized 6 nm gold nanoparticles (GNPs) to the cell wall polymers on poplar stem sections. The GNP-CBM3a complexes were found to bind to the cellulose surface, closely aligning along the cellulose fibril axis. The binding details were further confirmed and studied by single-molecule recognition imaging and AFM single-molecule dynamic force spectroscopy (SMDFS) using a CBM3a-functionalized AFM tip. The unbinding force was measured to be 44.96 ± 18.80 pN under a loading rate of 67.2 nN/s. This research provides a radical method for the study of single-molecule affinity between CBM and cellulose that is critical to the engineering of novel cellulolytic enzymes.

Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin

Protein interactions with peptides generally have low thermodynamic and mechanical stability. Streptococcus pyogenes fibronectin-binding protein FbaB contains a domain with a spontaneous isopeptide bond between Lys and Asp. By splitting this domain and rational engineering of the fragments, we obtained a peptide (SpyTag) which formed an amide bond to its protein partner (SpyCatcher) in minutes. Reaction occurred in high yield simply upon mixing and amidst diverse conditions of pH, temperature, and buffer. SpyTag could be fused at either terminus or internally and reacted specifically at the mammalian cell surface. Peptide binding was not reversed by boiling or competing peptide. Single-molecule dynamic force spectroscopy showed that SpyTag did not separate from SpyCatcher until the force exceeded 1 nN, where covalent bonds snap. The robust reaction conditions and irreversible linkage of SpyTag shed light on spontaneous isopeptide bond formation and should provide a targetable lock in cells and a stable module for new protein architectures.

Immobilizing a single DNA molecule at the apex of AFM tips through picking and ligation

A proper surface treatment of the substrate and the AFM tip with a structured molecule provided a new approach to pick a single DNA molecule with reasonable success rate (75%) by AFM. Picking a single molecule by AFM tip and realizing the single interaction will likely be useful for single molecule dynamic force spectroscopy, because it avoids the formation of problematic multiple interactions. As an extension, a new single DNA was allowed to hybridize with the picked DNA, and conjugated with the picker DNA by use of a ligase. The attachment of a single DNA at the apex was confirmed by measuring the force between the new DNA and the complementary DNA on the generic surface. Various applications of the tip for manipulating single molecules and preparing new nanomaterials are envisaged.

Substrate Binding Tunes Conformational Flexibility and Kinetic Stability of an Amino Acid Antiporter

We used single molecule dynamic force spectroscopy to unfold individual serine/threonine antiporters SteT from Bacillus subtilis. The unfolding force patterns revealed interactions and energy barriers that stabilized structural segments of SteT. Substrate binding did not establish strong localized interactions but appeared to be facilitated by the formation of weak interactions with several structural segments. Upon substrate binding, all energy barriers of the antiporter changed thereby describing the transition from brittle mechanical properties of SteT in the unbound state to structurally flexible conformations in the substrate-bound state. The lifetime of the unbound state was much shorter than that of the substrate-bound state. This leads to the conclusion that the unbound state of SteT shows a reduced conformational flexibility to facilitate specific substrate binding and a reduced kinetic stability to enable rapid switching to the bound state. In contrast, the bound state of SteT showed an increased conformational flexibility and kinetic stability such as required to enable transport of substrate across the cell membrane. This result supports the working model of antiporters in which alternate substrate access from one to the other membrane surface occurs in the substrate-bound state.

Single‐Molecule Approach to Understanding Multivalent Binding Kinetics

Multivalency results from the simultaneous binding of multiple ligands with multiple receptors. Understanding the effect of multivalency on binding kinetics in molecular and cellular systems may aid the development of new types of therapeutics or countermeasures to pathogen infection. Here, we describe a method using single-molecule dynamic force spectroscopy to determine the binding strength of antibody-protein complexes as a function of binding valency in a direct and simple measurement. We used the atomic force microscope to measure the force required to rupture a single complex formed by the MUC1 protein, a cancer indicator, and therapeutic antibodies that target MUC1. We will show that nanomechanical polymer tethers can be used in a new manner to count the number of biological bonds formed. Mechanical work will disrupt these bonds and can be used to quantify the overall kinetics. This ability to form, count, and dissociate biological bonds with nanomechanical forces provides a powerful method to study the physical laws governing the interactions of biological molecules.

A Simple and Practical Spreadsheet-Based Method to Extract Single-Molecule Dissociation Kinetics from Variable Loading-Rate Force Spectroscopy Data

A simple, practical, and model-free method is presented, by which kinetic data can be extracted from variable loading-rate single molecule dynamic force spectroscopy experiments. The constant sampling rate of digital acquisition facilitates the sorting of multiple force curves into histograms that reflect the collective force−time history of the bonds of interest. Combining the force−time history with similar histograms of force-induced events, in particular bond rupture, gives a direct measure of rate vs applied force. The utility of the method is demonstrated through its application to simulated data.

Role of Extracellular Glutamic Acids in the Stability and Energy Landscape of Bacteriorhodopsin

Bacteriorhodopsin (BR), a specialized nanomachine, converts light energy into a proton gradient to power Halobacterium salinarum. In this work, we analyze the mechanical stability of a BR triple mutant in which three key extracellular residues, Glu 9, Glu 194, and Glu 204, were mutated simultaneously to Gln. These three Glu residues are involved in a network of hydrogen bonds, in cation binding, and form part of the proton release pathway of BR. Changes in these features and the robust photocycle dynamics of wild-type (WT) BR are apparent when the three extracellular Glu residues are mutated to Gln. It is speculated that such functional changes of proteins go hand in hand with changes in their mechanical properties. Here, we apply single-molecule dynamic force spectroscopy to investigate how the Glu to Gln mutations change interactions, reaction pathways, and the energy barriers of the structural regions of WT BR. The altered heights and positions of individual energy barriers unravel the changes in the mechanical and the unfolding kinetic properties of the secondary structures of WT BR. These changes in the mechanical unfolding energy landscape cause the proton pump to choose unfolding pathways differently. We suggest that, in a similar manner, the changed mechanical properties of mutated BR alter the functional energy landscape favoring different reaction pathways in the light-induced proton pumping mechanism.

Analyzing Forced Unfolding of Protein Tandems by Ordered Variates, 2: Dependent Unfolding Times

Statistical analyses of forced unfolding data for protein tandems, i.e., unfolding forces (force-ramp) and unfolding times (force-clamp), used in single-molecule dynamic force spectroscopy rely on the assumption that the unfolding transitions of individual protein domains are independent (uncorrelated) and characterized, respectively, by identically distributed unfolding forces and unfolding times. In our previous work, we showed that in the experimentally accessible piconewton force range, this assumption, which holds at a lower constant force, may break at an elevated force level, i.e., the unfolding transitions may become correlated when force is increased. In this work, we develop much needed statistical tests for assessing the independence of the unobserved forced unfolding times for individual protein domains in the tandem and equality of their parent distributions, which are based solely on the observed ordered unfolding times. The use and performance of these tests are illustrated through the analysis of unfolding times for computer models of protein tandems. The proposed tests can be used in force-clamp atomic force microscopy experiments to obtain accurate information on protein forced unfolding and to probe data on the presence of interdomain interactions. The order statistics-based formalism is extended to cover the analysis of correlated unfolding transitions. The use of order statistics leads naturally to the development of new kinetic models, which describe the probabilities of ordered unfolding transitions rather than the populations of chemical species.

Mechanical Properties of Bovine Rhodopsin and Bacteriorhodopsin: Possible Roles in Folding and Function

Molecular interactions and mechanical properties that contribute to the stability and function of proteins are complex and of fundamental importance. In this study, we used single-molecule dynamic force spectroscopy (DFS) to explore the interactions and the unfolding energy landscape of bovine rhodopsin and bacteriorhodopsin. An analysis of the experimental data enabled the extraction of parameters that provided insights into the kinetic stability and mechanical properties of these membrane proteins. Individual structural segments of rhodopsin and bacteriorhodopsin have different properties. A core of rigid structural segments was observed in rhodopsin but not in bacteriorhodopsin. This core may reflect differences in mechanisms of protein folding between the two membrane proteins. The different structural rigidity of the two proteins may also reflect their adaptation to differing functions.