Single Molecule AFM Force Spectroscopy Research Articles

Single-Molecule Adhesion of a Stimuli-Responsive Oligo(ethylene glycol) Copolymer to Gold

Adhesion of environmentally responsive polymers to biocompatible surfaces is an important issue that has been explored in several nanobiotechnology applications. Here, we prepared multi-responsive statistical copolymers of two oligo(ethylene glycol) methyl ether methacrylate macromonomers with differing ethylene glycol side chain lengths using RAFT polymerization. The lower critical solution temperature of the copolymers was characterized using visible light extinction, and the chemical composition and molecular weight were measured using NMR spectroscopy and size-exclusion chromatography, respectively. The characterization results demonstrated that the transition temperature could be controlled by varying the macromonomer feed ratios, and the molecular weight could be controlled by varying the amount of the RAFT chain transfer agent in the polymerization feed. Using AFM single-molecule force spectroscopy, we measured the adhesion characteristics of single copolymer molecules to a gold surface. We found that dehydration and collapse of the copolymer in a high ionic strength buffer resulted in dramatically reduced bridging length distributions that maintained their single-molecule bimodal character. In the collapsed state, the polymer exhibited a lower absolute desorption force while cooperativity effects were found to increase the desorption force per chain for multi-chain interactions. Our results confirmed that the polymer in a collapsed conformation exhibited a dramatically reduced volume occupancy above the gold surface. These results demonstrate at the single-molecule level how solvent-induced collapse of an environmentally responsive copolymer modulates surface adhesion forces and bridging length distributions in a controllable way.

Effect of Spermidine on Misfolding and Interactions of Alpha-Synuclein

Alpha-synuclein (α-Syn) is a 140 aa presynaptic protein which belongs to a group of natively unfolded proteins that are unstructured in aqueous solutions. The aggregation rate of α-Syn is accelerated in the presence of physiological levels of cellular polyamines. Here we applied single molecule AFM force spectroscopy to characterize the effect of spermidine on the very first stages of α-Syn aggregation – misfolding and assembly into dimers. Two α-Syn variants, the wild-type (WT) protein and A30P, were studied. The two protein molecules were covalently immobilized at the C-terminus, one at the AFM tip and the other on the substrate, and intermolecular interactions between the two molecules were measured by multiple approach-retraction cycles. At conditions close to physiological ones at which α-Syn misfolding is a rare event, the addition of spermidine leads to a dramatic increase in the propensity of the WT and mutant proteins to misfold. Importantly, misfolding is characterized by a set of conformations, and A30P changes the misfolding pattern as well as the strength of the intermolecular interactions. Together with the fact that spermidine facilitates late stages of α-Syn aggregation, our data demonstrate that spermidine promotes the very early stages of protein aggregation including α-Syn misfolding and dimerization. This finding suggests that increased levels of spermidine and potentially other polyamines can initiate the disease-related process of α-Syn.

Effects of Electrostatics on Interactions of Short Peptide from Yeast Prion Protein Sup35

Protein misfolding and subsequent aggregation are the first steps in a long, complex process central to prion diseases. Previous experiments show distinct growth patterns at different pH values, with the fastest kinetics observed at pH 5.6, which is in line with the AFM force spectroscopy studies revealing the strongest interactions at pH 5.6. Since this pH corresponds to the peptide isoelectric point, we hypothesize that the observed effects are due to electrostatic interactions.Here, we studied the effect of electrostatics on the aggregation and peptide-peptide interactions of a short fragment of the yeast prion protein, Sup35 (GNNQQNY), used as a model to describe protein misfolding. We characterized aggregate growth using AFM and Thioflavin T (ThT) fluorescence measurements in various pHs and ionic strengths. We used single molecule AFM force spectroscopy to characterize peptide-peptide interactions at different ionic strengths. The electrostatic effect should be the strongest at pH values far from the pI and the lowest at the isoelectric point. Indeed, the experiments showed that at pH 2.0, the aggregation lag time is 195 hours under low ionic strength (10 mM) whereas the lag time decreases to 42 hours when ionic strength at pH 2.0 increases to 150 mM. At pH 5.6, the aggregation lag time is 11 hours under low ionic strength, and there is minimal change to lag time when ionic strength is increased. In parallel, AFM force spectroscopy studies were performed. They revealed minimal contribution of electrostatics in the dissociation of transient dimers. Increasing ionic strength at pH 2.0 resulted in a significant increase in forces. Such an increase in the magnitudes of rupture forces indicates that ionic strength enhanced inter-peptide interactions at low pH. The effect was less noticeable near the isoelectric point.

Functional characterization of a supramolecular affinity switch at the single molecule level

Surface-immobilized and switchable resorcin[4]arene receptor molecules were quantitatively investigated with atomic force microscopy (AFM) and AFM-single molecule force spectroscopy (AFM-SMFS). The upper rim of the supramolecular receptor cavities was modified with two photodimerizable anthracene moieties. The molecular constitution can be externally switched and controlled by exposure to ultraviolet (UV) light and heat. The topography as well as the complexation affinity against small ammonium guest ions of the two isomers were investigated at the single molecule level. Our results demonstrate the feasibility to externally control the supramolecular receptor's affinity and simultaneously quantify and associate these binding properties with the structural change of the resorcin[4]arenes structure on the basis of the measured molecule corrugation height.

Electrostatic Switching of Polysaccharide Conformation Probed at the Single Molecule Level

Polysaccharides play a key mechanical role in maintaining cell integrity and in cell-cell recognition. Single molecule AFM stretching measurements have revealed that upon loading, the backbone of polysaccharide molecules change their conformation and these conformational changes depend on the linkages between the sugar rings. It has been proposed that these force-induced conformational transitions may play an important role in biological systems. However a mechanism to switch on/off these conformational transitions and control the nanomechanical properties of carbohydrates has not yet been shown.Here we demonstrate an electrostatic switch that can be used to toggle the force dependent conformational transition in acidic polysaccharides. Using single molecule AFM force spectroscopy we show that the tension dependent conformation of the polysaccharide molecules can be controlled by varying the backbone charge density and solution electrostatics.

Misfolding and Aggregation of Amyloid Beta Peptide: Single Molecule AFM Force Spectroscopy

Misfolding and aggregation of amyloid beta (Aβ) peptide result in development of Alzheimer's disease, and Aβ dimers are considered as the smallest neurotoxic species. The aggregates formed by Aβ-peptides have been characterized by various techniques, but our knowledge on the molecular mechanism underlying the processes of misfolding and the early stages of aggregation of the peptides is limited. We have shown earlier that AFM force spectroscopy is capable of detecting protein misfolded states and characterizing the initial stages of the protein aggregation. Importantly, we showed that α-synuclein dimers are stable transient states playing role of triggers in the process of the protein self-assembly in nanopartiles and fibrils. Here we applied the sample methodology to probing and characterizing of misfolding of Aβ40 peptide. The protein was immobilized on the AFM tip and the surface and the interaction between the proteins was measured in multiple approach-retraction cycles. Using this approach was able to analyze interprotein interactions at single molecule level. The force spectroscopy analysis provided us with the following important information. First, using Dynamic Force Spectroscopy (DFS) approach we characterized pathways of Aβ40 misfolding. The lifetimes of transient Aβ40 dimers can be as long as several seconds suggesting that formation of the states with such a lifetimes can trigger the aggregation. Second, the pathways for the misfolding and aggregation depend on pH leading to a rather complex energy landscape reconstructed from the DFS data. Third, the analysis of the contour lengths supported the conclusions on various aggregation pathways and led to the models for such conformations of Aβ40.This work is supported by DE-FG02-08ER64579 grant from DOE.

Probing DNA–peptide interaction forces at the single‐molecule level

The versatility of chemical peptide synthesis combined with the high sensitivity of AFM single-molecule force spectroscopy allows us to investigate, quantify, and control molecular recognition processes (molecular nanotechnology), offering a tremendous potential in chemical biology.Single-molecule force spectroscopy experiments are able to detect fast intermediate transition states, details of the energy landscape, and structural changes, while avoiding multiple binding events that can occur under ensemble conditions. Dynamic force spectroscopy (DFS) is even able to provide data on the complex lifetime. This minireview outlines the biophysical methodology, discusses different experimental set-ups, and presents representative results in the form of two case studies, both dealing with DNA-binding peptides. They may serve as model systems, e.g., for transcription factors or gene transfection agents.

Single-molecule Force Spectroscopy Measurements of “Hydrophobic Bond” between Tethered Hexadecane Molecules

The hydrophobic effect is important for many biological and technological processes. Despite progress in theory, experimental data clarifying water structure and the interaction between hydrophobic solutes at the nanometer scale are scarce due to the very low solubility of hydrophobic species. This article describes an AFM single molecule force spectroscopy method to probe the interaction between molecules with low solubility and reports measurements of the strength and the length scale of the "hydrophobic bond" between hexadecane molecules. Hexadecane molecules are tethered by flexible poly(ethylene glycol) linkers to AFM probes and substrates, removing the aggregation state uncertainty of solution-based approaches as well as spurious surface effects. A shorter hydrophilic polymer layer is added to increase the accessibility of hydrophobic molecules for the force spectroscopy measurements. Statistical analysis of the rupture forces yields a barrier width of 0.24 nm, and a dissociation rate of 1.1 s(-1). The results of single molecule measurements are related to the theoretical predictions of the free energy of cavitation in water and to the empirical model of micellization of nonionic surfactants. It is estimated that approximately one-quarter of each molecule's surface is hydrated during forced dissociation, consistent with an extended (nonglobular) conformation of the hexadecane molecules in the dimer.

Mechanical properties of cardiac titin’s N2B-region by single-molecule atomic force spectroscopy

Titin is a giant protein responsible for passive-tension generation in muscle sarcomeres. Here, we used single-molecule AFM force spectroscopy to investigate the mechanical characteristics of a recombinant construct from the human cardiac-specific N2B-region, which harbors a 572-residue unique sequence flanked by two immunoglobulin (Ig) domains on either side. Force-extension curves of the N2B-construct revealed mean unfolding forces for the Ig-domains similar to those of a recombinant fragment from the distal Ig-region in titin (I91–98). The mean contour length of the N2B-unique sequence was 120 nm, but there was a bimodal distribution centered at ∼95 nm (major peak) and 180 nm (minor peak). These values are lower than expected if the N2B-unique sequence were a permanently unfolded entropic spring, but are consistent with the ∼100 nm maximum extension of that segment measured in isolated stretched cardiomyofibrils. A contour-length below 200 nm would be reasonable, however, if the N2B-unique sequence were stabilized by a disulphide bridge, as suggested by several disulphide connectivity prediction algorithms. Since the N2B-unique sequence can be phosphorylated by protein kinase A (PKA), which lowers titin-based stiffness, we studied whether addition of PKA (+ATP) affects the mechanical properties of the N2B-construct, but found no changes. The softening effect of PKA on N2B-titin may require specific conditions/factors present inside the cardiomyocytes.