alpha-Synuclein misfolding: single molecule AFM force spectroscopy study

Protein misfolding and aggregation are the very first and critical steps in development of various neurodegenerative disorders, including Parkinson’s disease, induced by misfolding of α-synuclein. Thus, elucidating properties of proteins in misfolded states and understanding the mechanisms of their assembly into the disease prone aggregates are critical for the development of rational approaches to prevent protein misfolding-mediated pathologies. To accomplish this goal and as a first step to elucidate the mechanism of α-synuclein misfolding, we applied single-molecule force spectroscopy capable of detecting protein misfolding. We immobilized α-synuclein molecules at their C-termini at the atomic force microscope tips and substrate surfaces, and measured the interaction between the proteins by probing the microscope tip at various locations on the surface. Using this approach, we detected α-synuclein misfolded states by enhanced interprotein interaction. We used a dynamics force spectroscopy approach to measure such an important characteristic of dimers of misfolded α-synuclein as their lifetimes. We found that the dimer lifetimes are in the range of seconds and these values are much higher than the characteristics for the dynamics of the protein in monomeric state. These data show that compared to highly dynamic monomeric forms, α-synuclein dimers are much more stable and thus can serve as stable nuclei for the formation of multimeric and aggregated forms of α-synuclein. Importantly, two different lifetimes were observed for the dimers, suggesting that aggregation can follow different pathways that may lead to different aggregated morphologies of α-synuclein.     

Discriminating small molecule DNA binding modes by single molecule force spectroscopy

Drugs may interact with double stranded DNA via a variety of binding modes, each mode giving rise to a specific pharmacological function. Here we demonstrate the ability of single molecule force spectroscopy to discriminate between different interaction modes by measuring the mechanical properties of DNA and their modulation upon the binding of small molecules. Due to the unique topology of double stranded DNA and due to its base pair stacking pattern, DNA undergoes several well-characterised structural transitions upon stretching. We show that small molecule binding markedly affects these transitions in ways characteristic to the binding mode and that these effects can be detected at the level of an individual molecule. The minor groove binder berenil, the crosslinker cisplatin and the intercalator ethidium bromide are compared.     

DNA base pair resolution by single molecule force spectroscopy

The forces that hold complementary strands of DNA together in a double helix, and the role of base mismatches in these, are examined by single molecule force spectroscopy using an atomic force microscope (AFM). These forces are important when considering the binding of proteins to DNA, since these proteins often mechanically stretch the DNA during their action. In AFM measurement of forces, there is an inherent instrumental limitation that makes it difficult to compare results from different experimental runs. This is circumvented by using an oligonucleotide microarray, which allowed a direct comparison of the forces between perfectly matched short oligonucleotides and those containing a single or double mismatch. Through this greatly increased sensitivity, the force contribution of a single AT base pair was derived. The results indicate that the contribution to forces from the stacking interactions is more important than that from hydrogen bonding.     

Pulling geometry-induced errors in single molecule force spectroscopy measurements

In AFM-based single molecule force spectroscopy, it is tacitly assumed that the pulling direction coincides with the end-to-end vector of the molecule fragment being stretched. By systematically varying the position of the attachment point on the substrate relative to the AFM tip, we investigate empirically and theoretically the effect of the pulling geometry on force-extension characteristics of double-stranded DNA. We find that increasing the pulling angle can significantly lower the force of the characteristic overstretching transition and increase the width of the plateau feature beyond the canonical 70%. These effects, when neglected, can adversely affect the interpretation of measured force-extension relationships. We quantitatively evaluate force and extension errors originating from this "pulling angle effect" and stress the need to correct the pulling geometry when stretching rigid molecules with an AFM.     

An automated two-dimensional optical force clamp for single molecule studies

We constructed a next-generation optical trapping instrument to study the motility of single motor proteins, such as kinesin moving along a microtubule. The instrument can be operated as a two-dimensional force clamp, applying loads of fixed magnitude and direction to motor-coated microscopic beads moving in vitro. Flexibility and automation in experimental design are achieved by computer control of both the trap position, via acousto-optic deflectors, and the sample position, using a three-dimensional piezo stage. Each measurement is preceded by an initialization sequence, which includes adjustment of bead height relative to the coverslip using a variant of optical force microscopy (to +/-4 nm), a two-dimensional raster scan to calibrate position detector response, and adjustment of bead lateral position relative to the microtubule substrate (to +/-3 nm). During motor-driven movement, both the trap and stage are moved dynamically to apply constant force while keeping the trapped bead within the calibrated range of the detector. We present details of force clamp operation and preliminary data showing kinesin motor movement subject to diagonal and forward loads.     

Minimizing pulling geometry errors in atomic force microscope single molecule force spectroscopy

In atomic force microscopy-based single molecule force spectroscopy (AFM-SMFS), it is assumed that the pulling angle is negligible and that the force applied to the molecule is equivalent to the force measured by the instrument. Recent studies, however, have indicated that the pulling geometry errors can drastically alter the measured force-extension relationship of molecules. Here we describe a software-based alignment method that repositions the cantilever such that it is located directly above the molecule's substrate attachment site. By aligning the applied force with the measurement axis, the molecule is no longer undergoing combined loading, and the full force can be measured by the cantilever. Simulations and experimental results verify the ability of the alignment program to minimize pulling geometry errors in AFM-SMFS studies.     

Detection of metal binding sites on functional S-layer nanoarrays using single molecule force spectroscopy

Crystalline bacterial cell surface layers (S-layers) show the ability to recrystallize into highly regular pattern on solid supports. In this study, the genetically modified S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177, carrying a hexa-histidine tag (His₆-tag) at the C-terminus, was used to generate functionalized two-dimensional nanoarrays on a silicon surface. Atomic force microscopy (AFM) was applied to explore the topography and the functionality of the fused His₆-tags. The accessibility of the His₆-tags was demonstrated by in-situ anti-His-tag antibody binding to the functional S-layer array. The metal binding properties of the His₆-tag was investigated by single molecule force microscopy. For this purpose, newly developed tris–NTA was tethered to the AFM tips via a flexible polyethylene glycol (PEG) linker. The functionalized tips showed specific interactions with S-layer containing His₆-tags in the presence of nickel ions. Thus the His₆-tag is located at the outer surface of the S-layer and can be used for stable but reversible attachment of functional tris–NTA derivatives.     

Efficient unfolding pattern recognition in single molecule force spectroscopy data

Background

Single-molecule force spectroscopy (SMFS) is a technique that measures the force necessary to unfold a protein. SMFS experiments generate Force-Distance (F-D) curves. A statistical analysis of a set of F-D curves reveals different unfolding pathways. Information on protein structure, conformation, functional states, and inter- and intra-molecular interactions can be derived.

Results

In the present work, we propose a pattern recognition algorithm and apply our algorithm to datasets from SMFS experiments on the membrane protein bacterioRhodopsin (bR). We discuss the unfolding pathways found in bR, which are characterised by main peaks and side peaks. A main peak is the result of the pairwise unfolding of the transmembrane helices. In contrast, a side peak is an unfolding event in the alpha-helix or other secondary structural element. The algorithm is capable of detecting side peaks along with main peaks. Therefore, we can detect the individual unfolding pathway as the sequence of events labeled with their occurrences and co-occurrences special to bR's unfolding pathway. We find that side peaks do not co-occur with one another in curves as frequently as main peaks do, which may imply a synergistic effect occurring between helices. While main peaks co-occur as pairs in at least 50% of curves, the side peaks co-occur with one another in less than 10% of curves. Moreover, the algorithm runtime scales well as the dataset size increases.

Conclusions

Our algorithm satisfies the requirements of an automated methodology that combines high accuracy with efficiency in analyzing SMFS datasets. The algorithm tackles the force spectroscopy analysis bottleneck leading to more consistent and reproducible results.     

Molecular dynamics simulation of dextran extension by constant force in single molecule AFM

The extension of 1-6 polysaccharides has been studied in a series of recent single molecule AFM experiments. For dextran, a key finding was the existence of a plateau in the force-extension curve at forces between 700 and 1000 pN. We studied the extension of the dextran 10-mer under constant force using atomistic simulation with various force fields. All the force fields reproduce the experimental plateau on the force-extension curve. With AMBER94 and AMBER-GLYCAM04 force fields the plateau can be explained by a transition of the glucopyranose rings in the dextran monomers from the chair ((4)C(1)) to the inverted chair ((1)C(4)) conformation while other processes occur at smaller (rotation around C5-C6 bond) or higher (chairs to boat transitions) forces. The CHARMM force field provides a different picture which associates the occurrence of the plateau to chair-boat transitions of the glucopyranose rings.     

Single molecule force spectroscopy in biology using the atomic force microscope

The importance of forces in biology has been recognized for quite a while but only in the past decade have we acquired instrumentation and methodology to directly measure interactive forces at the level of single biological macromolecules and/or their complexes. This review focuses on force measurements performed with the atomic force microscope. A general introduction to the principle of action is followed by review of the types of interactions being studied, describing the main results and discussing the biological implications.     

Mechanistic insights into the physiological functions of cell adhesion proteins using single molecule force spectroscopy

Intercellular adhesion molecules play an important role in regulating several cellular processes such as a proliferation, migration and differentiation. They also play an important role in regulating solute diffusion across monolayers of cells. The adhesion characteristics of several intercellular adhesion molecules have been studied using various biochemical assays. However, the advent of single molecule force spectroscopy as a powerful tool to analyze the kinetics and strength of protein interactions has provided us with an opportunity to investigate these interactions at the level of a single molecule. The study of interactions involving intercellular adhesion molecules has gained importance because of the fact that qualitative and quantitative changes in these proteins are associated with several disease processes. In this review, we focus on the basic principles, data acquisition and analysis in single molecule force spectroscopy experiments. Furthermore, we discuss the correlation between results obtained using single molecule force experiments and the physiological functions of the proteins in the context of intercellular adhesion molecules. Finally, we summarize some of the diseases associated with changes in intercellular adhesion molecules.     

Characterization of enhanced monovalent and bivalent thrombin DNA aptamer binding using single molecule force spectroscopy

Thrombin aptamer binding strength and stability is dependent on sterical parameters when used for atomic force microscopy sensing applications. Sterical improvements on the linker chemistry were developed for high-affinity binding. For this we applied single molecule force spectroscopy using two enhanced biotinylated thrombin aptamers, BFF and BFA immobilized on the atomic force microscopy tip via streptavidin. BFF is a dimer composed of two single-stranded aptamers (aptabody) connected to each other by a complementary sequence close to the biotinylated end. In contrast, BFA consists of a single DNA strand and a complementary strand in the supporting biotinylated part. By varying the pulling velocity in force-distance cycles the formed thrombin-aptamer complexes were ruptured at different force loadings allowing determination of the energy landscape. As a result, BFA aptamer showed a higher binding force at the investigated loading rates and a significantly lower dissociation rate constant, k_off, compared to BFF. Moreover, the potential of the aptabody BFF to form a bivalent complex could clearly be demonstrated.     

Covalent immobilization of recombinant fusion proteins with hAGT for single molecule force spectroscopy

A genetically modified form of the human DNA repair protein O⁶-alkylguanine-DNA-alkyltransferase (hAGT) was used to immobilize different recombinant hAGT fusion proteins covalently and selectively on gold and glass surfaces. Fusion proteins of hAGT with Glutathione S-Transferase and with tandem repeats of Titin Ig-domains, were produced and anchored via amino-polyethylene glycol benzylguanine. Anchoring was characterized and quantified with surface plasmon resonance, atomic force microscope and fluorescence measurements. Individual fusion proteins were unfolded by single molecule force spectroscopy corroborating the selectivity of the covalent attachment.     

Unequivocal single-molecule force spectroscopy of proteins by AFM using pFS vectors

Nanomechanical analysis of proteins by single-molecule force spectroscopy based on atomic force microscopy is increasingly being used to investigate the inner workings of mechanical proteins and substrate proteins of unfoldase machines as well as to gain new insight into the process of protein folding. However, such studies are hindered by a number of technical problems, including the noise of the proximal region, ambiguous single-molecule identification, as well as difficulties in protein expression/folding and full-length purification. To overcome these major drawbacks in protein nanomechanics, we designed a family of cloning/expression vectors, termed pFS (plasmid for force spectroscopy), that essentially has an unstructured region to surmount the noisy proximal region, a homomeric polyprotein marker, a carrier to mechanically protect the protein of interest (only the pFS-2 version) that also acts as a reporter, and two purification tags. pFS-2 enables the unambiguous analysis of proteins with low mechanical stability or/and complex force spectra, such as the increasingly abundant class of intrinsically disordered proteins, which are hard to characterize by traditional bulk techniques and have important biological and clinical implications. The advantages, applications, and potential of this ready-to-go system are illustrated through the analysis of representative proteins.     

Nonspecific interactions in AFM force spectroscopy measurements

Sample-probe contact duration (dwell time) and loading force are two important parameters for the atomic force microscopy (AFM) force spectroscopy measurements of ligand-receptor interaction. A prolonged contact time may be required to initiate ligand-receptor binding as a result of slow on-rate kinetics or low reactant density. In general, increasing contact duration promotes nonspecific interactions between the substrate and the functionalized cantilever and, thus, masking the detection of the specific interactions. To reduce the nonspecific interactions in AFM force measurements requiring extended substrate-probe contact, we investigated the interaction of bovine serum albumin (BSA)-functionalized cantilever with BSA-coated glass, polyethylene glycol (PEG)-functionalized glass, Pluronic-treated Petri dishes and agarose beads. The frequency of nonspecific interaction between the BSA-functionalized cantilever and the different samples increased with loading force and dwell time. This increase in nonspecific adhesion can be attributed to the interaction mediated by forced unfolding of BSA. By reducing the loading force, the contact duration of the AFM probe with an agarose bead can be extended to a few minutes without nonspecific adhesion.     

Efficient golden gate assembly of DNA constructs for single molecule force spectroscopy and imaging

Single-molecule techniques such as optical tweezers and fluorescence imaging are powerful tools for probing the biophysics of DNA and DNA-protein interactions. The application of these methods requires efficient approaches for creating designed DNA structures with labels for binding to a surface or microscopic beads. In this paper, we develop a simple and fast technique for making a diverse range of such DNA constructs by combining PCR amplicons and synthetic oligonucleotides using golden gate assembly rules. We demonstrate high yield fabrication of torsionally-constrained duplex DNA up to 10 kbp in length and a variety of DNA hairpin structures. We also show how tethering to a cross-linked antibody substrate significantly enhances measurement lifetime under high force. This rapid and adaptable fabrication method streamlines the assembly of DNA constructs for single molecule biophysics.     

Unfolding mechanics of multiple OspA substructures investigated with single molecule force spectroscopy

We investigated mechanical unfolding of Borrelia burgdorferi outer surface protein A (OspA), a Lyme disease antigen containing a unique single-layer beta-sheet, with atomic force microscopy (AFM). We mechanically stretched a monomeric unit, rather than a tandem repeat, by pulling it from its N and C-terminal residues without using intervening polymer as a spacer. We detected two peaks in the force-extension profile before the final rupture of a fully extended polypeptide, which we interpreted as unfolding of multiple substructures in OspA. The double-peaked unfolding curves are consistent with results of previous thermodynamic studies showing two cooperative units in OspA. The mechanical unfolding processes were reversible, and the two substructures refolded within one second. Mutations near the boundary of the two thermodynamic cooperative units reduced the height of the first unfolding peak to undetectable levels and marginally affected the second one, indicating that the boundary between the two mechanical substructures is related to that previously assigned between the thermodynamic cooperative units. Based on a "worm-like chain" analysis of our AFM data, we propose a model for mechanical unfolding of OspA, where nearly a half of the chain is stretched with minimal resistive force, followed by sequential breakdown of C-terminal and N-terminal substructures. Based on these results, we discuss similarities and differences between mechanical and thermodynamic unfolding reactions of OspA. This work demonstrates that AFM study of monomeric proteins can elucidate details of the intramolecular mechanics of protein substructures.     

40 Probing single molecule RNA folding using temperature and force

Nucleic acids can be unfolded either by temperature, such as in UV melting, or by mechanical force using optical tweezers. In UV melting experiments, the folding free energy of nucleic acids at mesophilic temperatures are extrapolated from unfolding occurring at elevated temperatures. Additionally, single molecule unfolding experiments are typically performed only at room temperature, preventing calculation of changes in enthalpy and entropy. Here, we present temperature-controlled optical tweezers suitable for studying folding of single RNA molecules at physiological temperatures. Constant temperatures between 22 and 37?°C are maintained with an accuracy of 0.1?°C, whereas the optical tweezers display a spatial resolution of ～1?nm over the temperature range. Using this instrument, we measured the folding thermodynamics and kinetics of a 20-base-pair RNA hairpin by force-ramp and constant force experiments. Between 22 and 37?°C, the hairpin unfolds and refolds in a single step. Increasing temperature decreases the stability of the hairpin and thus decreases the force required to unfold it. The equilibrium force, at which unfolding and refolding rates are equal, drops ～1?pN as temperature increases every 5?°C. At each temperature, the folding energy can be quantified by reversible work done to unfold the RNA and from the equilibrium constant at constant forces. Over the experimental temperature range, the folding free energy of the hairpin depends linearly on temperature, indicating that ΔH is constant. The measured folding thermodynamics are further compared with the nearest neighbor calculations using Turner’s parameters of nucleic acid folding energetics.