Single-molecule Force Spectroscopy Experiments Research Articles

Dynamic AFM force spectroscopy of DNA using FM mode with constant excitation

We report single-molecule force spectroscopy experiments on double-stranded DNA using a dynamically oscillating AFM cantilever driven in a frequency modulation mode with a constant excitation (CE) amplitude. While the DNA molecule is stretched, we measured the shift of the cantilever amplitude and the resonance frequency and estimated both the conservative forces and the dissipated energy. Typical elastic features of the calculated force distance curves are in nice agreement with data known from quasi-static stretching experiments. Analysis of the data indicates that the melting dynamics of the DNA base pairs depends on the amount of already melted base pairs and the base pair recombination rate. Furthermore, we observe a reduction of the molecular elasticity modulus, which is most likely caused by the cantilever dynamics leading to a destabilization of stacking and hydrogen bond interactions.

Structural and Mechanistic Insights into the Copper-Modulated Unfolding Pathways of Azurin

Characterization of energy landscape of metalloproteins is challenging because of the subtle changes imparted by metal binding on the stability and conformational flexibility of proteins. Here we present copper-binding induced changes in the mechanical unfolding pathways of azurin, a β-barrel protein with type-I copper center, through a joint experimental-computational study. Single-molecule force spectroscopy experiments reveal an intermediate along the unfolding pathway of apo-azurin in half of population. Steered molecular dynamics simulations attribute the native and intermediate unfolding transition states (TSs) to the rupture of interactions between the pairs of β-strands, 2B-8 and 4-7, respectively. We show that the copper-binding does not change the first TS of azurin and its mechanical properties, but influences the second TS. The rupture of 4-7 β-strand pair is delayed in holo-azurin because of constraints imposed by the copper coordination sphere and occurs after the second TS. Our experimental and computational approach extracts unprecedented details along the unfolding landscape for apo- and holo- forms of azurin.

Single‐Molecule Force Spectroscopy Trajectories of a Single Protein and Its Polyproteins Are Equivalent: A Direct Experimental Validation Based on A Small Protein NuG2

Single-molecule force spectroscopy (SMFS) has become a powerful tool in investigating the mechanical unfolding/folding of proteins at the single-molecule level. Polyproteins made of tandem identical repeats have been widely used in atomic force microscopy (AFM)-based SMFS studies, where polyproteins not only serve as fingerprints to identify single-molecule stretching events, but may also improve statistics of data collection. However, the inherent assumption of such experiments is that all the domains in the polyprotein are equivalent and one SMFS trajectory of stretching a polyprotein made of n domains is equivalent to n trajectories of stretching a single domain. Such an assumption has not been validated experimentally. Using a small protein NuG2 and its polyprotein (NuG2)4 as model systems, here we use optical trapping (OT) to directly validate this assumption. Our results show that OT experiments on NuG2 and (NuG2)4 lead to identical parameters describing the unfolding and folding kinetics of NuG2, demonstrating that indeed stretching a polyprotein of NuG2 is equivalent to stretching single NuG2 in force spectroscopy experiments and thus validating the use of polyproteins in SMFS experiments.

Hydration Effects Turn a Highly Stretched Polymer from an Entropic into an Energetic Spring.

Polyethylene glycol (PEG) is a structurally simple and nontoxic water-soluble polymer that is widely used in medical and pharmaceutical applications as molecular linker and spacer. In such applications, PEG's elastic response against conformational deformations is key to its function. According to text-book knowledge, a polymer reacts to the stretching of its end-to-end separation by a decrease in entropy that is due to the reduction of available conformations, which is why polymers are commonly called entropic springs. By a combination of single-molecule force spectroscopy experiments with molecular dynamics simulations in explicit water, we show that entropic hydration effects almost exactly compensate the chain conformational entropy loss at high stretching. Our simulations reveal that this entropic compensation is due to the stretching-induced release of water molecules that in the relaxed state form double hydrogen bonds with PEG. As a consequence, the stretching response of PEG is predominantly of energetic, not of entropic, origin at high forces and caused by hydration effects, while PEG backbone deformations only play a minor role. These findings demonstrate the importance of hydration for the mechanics of macromolecules and constitute a case example that sheds light on the antagonistic interplay of conformational and hydration degrees of freedom.

Structural Heterogeneity of CNGA1 Channels Revealed by Electrophysiology and Single-Molecule Force Spectroscopy.

The determination at atomic resolution of the three-dimensional molecular structure of membrane proteins such as receptors and several ion channels has been a major breakthrough in structural biology. The molecular structure of several members of the superfamily of voltage-gated ionic channels such as K+ and Na+ is now available. However, despite several attempts, the molecular structure at atomic resolution of the full cyclic nucleotide-gated (CNG) ion channel, although a member of the same superfamily of voltage-gated ion channels, has not been obtained yet, neither by X-ray crystallography nor by electron cryomicroscopy (cryo-EM). It is possible that CNG channels have a high structural heterogeneity, making difficult crystallization and single-particle analysis. To address this issue, we have combined single-molecule force spectroscopy (SMFS) and electrophysiological experiments to characterize the structural heterogeneity of CNGA1 channels expressed in Xenopus laevis oocytes. The unfolding of the cytoplasmic domain had force peaks, occurring with a probability from 0.2 to 0.96. Force peaks during the unfolding of the transmembrane domain had a probability close to 1, but the distribution of the increase in contour length between two successive force peaks had multiple maxima differing by tens of nanometers. Concomitant electrophysiological experiments showed that the rundown in mutant channels S399C is highly variable and that the effect of thiol reagents when specific residues were mutated was consistent with a dynamic structural heterogeneity. These results show that CNGA1 channels have a wide spectrum of native conformations that are difficult to detect with X-ray crystallography and cryo-EM.

A Multiscale Investigation on the Mechanism of Shape Recovery for IPDI to PPDI Hard Segment Substitution in Polyurethane

Shape memory thermoplastic polyurethane (SMTPU) containing isophorone diisocyanate (IPDI) in hard segment has excellent shape recoverability even after large strain deformation. However, the underlying mechanism of shape recovery remains unclear. In this study, 1,4-phenylene diisocyanate (PPDI) in the polyurethane is gradually substituted by IPDI, and multiscale effects are examined by normal and dichroic Fourier transform infrared spectroscopy (FTIR), small-angle X-ray scattering (SAXS), single-molecule force spectroscopy (SMFS), and mechanical test. Contradictory to the traditional conclusion, the degree of microphase separation decreases as the content of IPDI increases, while the macroscopic shape recoverability is largely improved. With dichroic FTIR and SAXS, we find that the morphology of hard phases changed from lamellar-like to fibrillar-like, which is more stable under stretching. SMFS experiments discover that IPDI could increase the elasticity of polymer chain and could endow the hard phases w...

Approach to the unfolding and folding dynamics of add A-riboswitch upon adenine dissociation using a coarse-grained elastic network model.

Riboswitches are noncoding mRNA segments that can regulate the gene expression via altering their structures in response to specific metabolite binding. We proposed a coarse-grained Gaussian network model (GNM) to examine the unfolding and folding dynamics of adenosine deaminase (add) A-riboswitch upon the adenine dissociation, in which the RNA is modeled by a nucleotide chain with interaction networks formed by connecting adjoining atomic contacts. It was shown that the adenine binding is critical to the folding of the add A-riboswitch while the removal of the ligand can result in drastic increase of the thermodynamic fluctuations especially in the junction regions between helix domains. Under the assumption that the native contacts with the highest thermodynamic fluctuations break first, the iterative GNM simulations showed that the unfolding process of the adenine-free add A-riboswitch starts with the denature of the terminal helix stem, followed by the loops and junctions involving ligand binding pocket, and then the central helix domains. Despite the simplified coarse-grained modeling, the unfolding dynamics and pathways are shown in close agreement with the results from atomic-level MD simulations and the NMR and single-molecule force spectroscopy experiments. Overall, the study demonstrates a new avenue to investigate the binding and folding dynamics of add A-riboswitch molecule which can be readily extended for other RNA molecules.

Mutual A domain interactions in the force sensing protein von Willebrand factor.

The von Willebrand factor (VWF) is a glycoprotein in the blood that plays a central role in hemostasis. Among other functions, VWF is responsible for platelet adhesion at sites of injury via its A1 domain. Its adjacent VWF domain A2 exposes a cleavage site under shear to degrade long VWF fibers in order to prevent thrombosis. Recently, it has been shown that VWF A1/A2 interactions inhibit the binding of platelets to VWF domain A1 in a force-dependent manner prior to A2 cleavage. However, whether and how this interaction also takes place in longer VWF fragments as well as the strength of this interaction in the light of typical elongation forces imposed by the shear flow of blood remained elusive. Here, we addressed these questions by using single molecule force spectroscopy (SMFS), Brownian dynamics (BD), and molecular dynamics (MD) simulations. Our SMFS measurements demonstrate that the A2 domain has the ability to bind not only to single A1 domains but also to VWF A1A2 fragments. SMFS experiments of a mutant [A2] domain, containing a disulfide bond which stabilizes the domain against unfolding, enhanced A1 binding. This observation suggests that the mutant adopts a more stable conformation for binding to A1. We found intermolecular A1/A2 interactions to be preferred over intramolecular A1/A2 interactions. Our data are also consistent with the existence of two cooperatively acting binding sites for A2 in the A1 domain. Our SMFS measurements revealed a slip-bond behavior for the A1/A2 interaction and their lifetimes were estimated for forces acting on VWF multimers at physiological shear rates using BD simulations. Complementary fitting of AFM rupture forces in the MD simulation range adequately reproduced the force response of the A1/A2 complex spanning a wide range of loading rates. In conclusion, we here characterized the auto-inhibitory mechanism of the intramolecular A1/A2 bond as a shear dependent safeguard of VWF, which prevents the interaction of VWF with platelets.

Single-molecule force spectroscopy on polyproteins and receptor–ligand complexes: The current toolbox

Single-molecule force spectroscopy sheds light onto the free energy landscapes governing protein folding and molecular recognition. Since only a single molecule or single molecular complex is probed at any given point in time, the technique is capable of identifying low-probability conformations within a large ensemble of possibilities. It furthermore allows choosing certain unbinding pathways through careful selection of the points at which the force acts on the protein or molecular complex. This review focuses on recent innovations in construct design, site-specific bioconjugation, measurement techniques, instrumental advances, and data analysis methods for improving workflow, throughput, and data yield of AFM-based single-molecule force spectroscopy experiments. Current trends that we highlight include customized fingerprint domains, peptide tags for site-specific covalent surface attachment, and polyproteins that are formed through mechanostable receptor-ligand interactions. Recent methods to improve measurement stability, signal-to-noise ratio, and force precision are presented, and theoretical considerations, analysis methods, and algorithms for analyzing large numbers of force-extension curves are further discussed. The various innovations identified here will serve as a starting point to researchers in the field looking for opportunities to push the limits of the technique further.

Examining the Mechanical Properties of Copper Binding Azurin using Single Molecule Force Spectroscopy and Steered Molecular Dynamics

The nature and properties of chemical bonds or interactions are of fundamental importance in understanding reactivity as well as biological functions of proteins. In particular, the metal-ligand bonds in metalloproteins are of special interest due to their partial covalent character and redox properties which might be sensitive to the protein environment. Hence, it is important to study and compare their mechanical strength and stability with those of covalent bonds. Here, we have investigated the mechanical properties of copper binding azurin using single molecule force spectroscopy (SMFS) and compared them with those of apo azurin. Azurin is a periplasmic protein which undergoes forced-unfolding during translocation from cytoplasm to periplasm. Our SMFS pulling studies on heptamer of azurin have revealed that holo azurin (copper bound protein) as well as its apo form unfold via an intermediate, which is mechanically weaker than the native state. We observed that the unfolding force of holo azurin is slightly higher (∼10 pN) than that of apo azurin. Steered molecular dynamic simulations (SMD) simulations on apo azurin showed that the unraveling is initiated from the C-terminus by the rupture of H-bonds between β8 and β2B strands. Solvent accessibility of the native disulfide bond of azurin in the X-ray structure and in MD simulation showed that only ∼15% of its surface area is accessible to the external aqueous solvent. Indeed, we have found from our pulling experiments that the native disulfide bond is inaccessible and resistant to chemical reduction by DTT and β-mercapto-ethanol. However, the disulfide bond gets exposed after the initial rupture of the native state and undergoes mechano-chemical reduction in SMFS experiments.

Mutual a Domain Interactions in the Force Sensing Protein von Willebrand Factor (VWF)

The von Willebrand factor (VWF) is a glycoprotein in the blood that plays a central role in hemostasis. Among other functions, VWF is responsible for platelet adhesion at sites of injury via its A1 domain. Its adjacent VWF domain A2 exposes a cleavage site under shear to degrade long VWF fibers in order to prevent thrombosis. Recently, it has been shown that VWF A1/A2 interactions inhibit the binding of platelets to VWF domain A1 in a force-dependent manner prior to A2 cleavage. However, whether and how this interaction also takes place in longer VWF fragments as well as the strength of this interaction in the light of typical elongation forces imposed by the shear flow of blood remained elusive. Here, we addressed these questions by using single molecule force spectroscopy (SMFS), Brownian dynamics (BD), and molecular dynamics (MD) simulations. Our SMFS measurements demonstrate that the A2 domain has the ability to bind not only to single A1 domains but also to VWF A1A2 fragments. SMFS experiments of a mutant [A2] domain, containing a disulfide bond which stabilizes the domain against unfolding, enhanced A1 binding. This observation suggests that the mutant adopts a more stable conformation for binding to A1. We found intermolecular A1/A2 interactions to be preferred over intramolecular A1/A2 interactions. Our data are also consistent with the existence of two cooperatively acting binding sites for A2 in the A1 domain. Our SMFS measurements revealed a slip-bond behavior for the A1/A2 interaction and their lifetimes were estimated for forces acting on VWF multimers at physiological shear rates using BD simulations. Complementary fitting of AFM rupture forces in the MD simulation range adequately reproduced the force response of the A1/A2 complex spanning a wide range of loading rates. In conclusion, we here characterized the auto-inhibitory mechanism of the intramolecular A1/A2 bond as a shear dependent safeguard of VWF, which prevents the interaction of VWF with platelets. 2016 The Authors. Published by Elsevier Inc. ThisisanopenaccessarticleundertheCCBY-NC-NDlicense

Folding and Catalysis of the glmS Ribozyme Riboswitch Studied at the Single-Molecule Level

We present findings from optical trapping experiments performed on the glmS ribozyme riboswitch. Found in the glmS gene in many bacteria, the glmS riboswitch down-regulates GlmS expression in the presence of the cell wall precursor—and enzymatic product of GlmS—glucosamine-6-phosphate (GlcN6P). In response to GlcN6P, the riboswitch site-specifically cleaves itself near its 5ʹ end, which targets the mRNA for subsequent degradation1. We performed single-molecule force spectroscopy experiments on the ribozyme catalytic core, unfolding and refolding the glmS RNA under controlled mechanical loads. The force-extension curves (FECs) reveal that folding and unfolding occur through a series of intermediate states, and we observe a striking hysteresis between unfolding and refolding of the ribozyme. Analysis of the FEC data leads to a model for the major unfolding pathway, involving the sequential unfolding of a series of discrete substructures. We’ve also begun to characterize the folding energy landscape of the catalytic core, using a series of constant-force measurements. We performed self-cleavage assays with optically trapped ribozyme molecules in the presence of the enzymatic cofactor GlcN6P and its catalytically inactive analog, glucose 6-phosphate. These assays demonstrate that our experimental construct, consisting of aminimal ribozyme sequence, is enzymatically active and strictly dependent upon the presence of GlcN6P. Self-cleavage measurements under controlled loads allow us to determine the profile of the catalytic activity in response to destabilizing forces. We observe an intermediate destabilization regime where the ribozyme remains active, but displays a partial loss of function.Footnotes1 Collins, J.A., Irnov, I., Baker, S., and Winkler, W.C. (2007) Genes Dev21(24), 3356-68.

Molecular Tools for Advanced Single-Molecule Studies

Specific attachment and tethering strategies are of utmost importance to study single biomolecules, e.g. in force and fluorescence spectroscopy. To this end we implement and improve molecular tools for use in e.g. AFM-based Single-Molecule Force Spectroscopy (SMFS).The biotin:(Strept)avidin interaction inevitably comes to mind, whenever detection, trapping or purification of biomolecules is desired. Derived thereof, the Strep-Tag II peptide/Strep-Tactin pair eliminates the necessity of biotin-labelling when working with protein specimens. As for (Strept)avidin, the tetravalency in Strep-Tactin is unfavourable for single-molecule applications. We therefore developed a monovalent Strep-Tactin featuring well-defined binding geometry and stoichiometry, yet unaltered affinity towards Strep-Tagged proteins. A unique Cysteine allows for selective immobilization or fluorescence labelling of this construct. We exploited the mechanical properties of the Strep-Tag II:monovalent Strep-Tactin interaction utilizing AFM-based single-molecule force spectroscopy. Rupture forces up to 200 pN are observed, which is comparable to biotin:(Strept)avidin unbinding. The applicability of the system in various force spectroscopy settings to study proteins of interest is evident. In addition, monovalent Strep-Tactin can be considered a precision tool that can replace (Strept)avidin in various applications, especially in mechanically demanding environments and when utilization of the genetically encoded Strep-Tag II is preferential to biotin-labelling.We utilize this novel tethering strategy to for example characterize force-sensing kinases, like Myosin Light Chain Kinase and Focal Adhesion Kinase in AFM SMFS experiments.

Adsorption orientations and immunological recognition of antibodies on graphene.

Large-scale molecular dynamics (MD) simulations and atomic force microscopy (AFM) in liquid are combined to characterize the adsorption of Immunoglobulin G (IgG) antibodies over a hydrophobic surface modeled with a three-layer graphene slab. We consider explicitly the water solvent, simulating systems with massive sizes (up to 770 000 atoms), for four different adsorption orientations. Protocols based on steered MD to speed up the protein diffusion stage and to enhance the dehydration process are combined with long simulation times (>150 ns) in order to make sure that the final adsorption states correspond to actual stable configurations. Our MD results and the AFM images demonstrate that the IgG antibodies are strongly adsorbed, do not unfold, and retain their secondary and tertiary structures upon deposition. Statistical analysis of the AFM images shows that many of the antibodies adopt vertical orientations, even at very small coverages, which expose at least one Fab binding site for recognition events. Single molecule force spectroscopy experiments demonstrate the immunological response of the deposited antibodies by recognizing its specific antigens. The above properties together with the strong anchoring and preservation of the secondary structure, make graphene an excellent candidate for the development of immunosensors.

Accelerating a Mechanically Driven anti-Woodward-Hoffmann Ring Opening with a Polymer Lever Arm Effect.

Mechanical forces have previously been used to drive reactions along pathways that violate the orbital symmetry effects captured in the Woodward-Hoffmann rules. Here, we show that a polymer "lever arm effect" can provide a mechanical advantage in accelerating the symmetry forbidden disrotatory ring opening of benzocyclobutene (BCB). Addition of an α-E-alkene to the BCB mechanophore drops the force required to induce reactions on the ∼0.1 s time scale of single-molecule force spectroscopy experiments from 1370 to 920 pN.

C-5 Propynyl Modifications Enhance the Mechanical Stability of DNA.

Increased thermal or mechanical stability of DNA duplexes is desired for many applications in nanotechnology or -medicine where DNA is used as a programmable building block. Modifications of pyrimidine bases are known to enhance thermal stability and have the advantage of standard base-pairing and easy integration during chemical DNA synthesis. Through single-molecule force spectroscopy experiments with atomic force microscopy and the molecular force assay we investigated the effect of pyrimidines harboring C-5 propynyl modifications on the mechanical stability of double-stranded DNA. Utilizing these complementary techniques, we show that propynyl bases significantly increase the mechanical stability if the DNA is annealed at high temperature. In contrast, modified DNA complexes formed at room temperature and short incubation times display the same stability as non-modified DNA duplexes.

Extraction of accurate biomolecular parameters from single-molecule force spectroscopy experiments.

The atomic force microscope (AFM) is able to manipulate biomolecules and their complexes with exquisite force sensitivity and distance resolution. This capability, complemented by theoretical models, has greatly improved our understanding of the determinants of mechanical strength in proteins and revealed the diverse effects of directional forces on the energy landscape of biomolecules. In unbinding experiments, the interacting partners are usually immobilized on their respective substrates via extensible linkers. These linkers affect both the force and contour length (Lc) of the complex at rupture. Surprisingly, while the former effect is well understood, the latter is largely neglected, leading to incorrect estimations of Lc, a parameter that is often used as evidence for the detection of specific interactions and remodeling events and for the inference of interaction regions. To address this problem, a model that predicts contour length measurements from single-molecule forced-dissociation experiments is presented that considers attachment position on the AFM tip, geometric effects, and polymer dynamics of the linkers. Modeled data are compared with measured contour length distributions from several different experimental systems, revealing that current methods underestimate contour lengths. The model enables nonspecific interactions to be identified unequivocally, allows accurate determination of Lc, and, by comparing experimental and modeled distributions, enables partial unfolding events before rupture to be identified unequivocally.

Mechanical Aspects of Protein Thermostability

Because thermophilic proteins are stable and functional at high temperature but generally lack activity under ambient conditions, it was suggested that thermal stability can correspond to enhanced mechanical rigidity. While this concept has initially found wide experimentally support, its generality has been later questioned. It is then natural to inquire the correlation between protein thermal and mechanical stabilities using single-molecule force-spectroscopy experiments. Here, we will present results of a combination of computational techniques, mainly based on Molecular Dynamics simulations, to tackle these problems for several mesophilic/thermophilic homologues of different molecular complexity.By applying innovative network analysis to map the protein conformational landscape, we first question the universality of the rigidity paradigm. In fact we show that for several paris of homologues, thermophiles can be as flexible as their mesophilic variant with important effects coming from the local partition of rigid/flexible regions, protein oligomerization state, or the time and length scales considered.We will then focus on the correlation among thermostability and mechanical resistance. The object under investigation is a pair of mesophilic/thermophilic cold-shock proteins for which experimental data are available. We have first implemented an enhanced-sampling algorithm which allows us to explore the proteins’ free-energy landscape over a wide range of temperatures, and we have used constant-force steered MD to investigate the effect of mechanical force. Our results suggest that thermal stability does not guarantee a mechanical one and that the associated unfolding mechanisms, of which our simulations provide extensive details, are very different. We also aim at identifying the weak-points of the protein mechanical stability and how more stable mutants can be designed. If such mutants also exhibit increased thermal stability, our approach could prove useful in numerous applications.

Small Peptide Binding Stiffens the Ubiquitin-like Protein SUMO1

Posttranslational modification by small ubiquitin-like modifiers (SUMOs), known as SUMOylation, is a key regulatory event in many eukaryotic cellular processes in which SUMOs interact with a large number of target proteins. SUMO binding motifs (SBMs) are small peptides derived from these target proteins that interact noncovalently with SUMOs and induce conformational changes. To determine the effect of SBMs on the mechanical properties of SUMO1 (the first member of the human SUMO family), we performed single-molecule force spectroscopy experiments on SUMO1/SBM complexes. The unfolding force of SUMO1 (at a pulling speed of 400 nm/s) increased from ∼130 pN to ∼170 pN upon binding to SBMs, indicating mechanical stabilization upon complexation. Pulling-speed-dependent experiments and Monte Carlo simulations measured a large decrease in distance to the unfolding transition state for SUMO1 upon SBM binding, which is by far the largest change measured for any ligand binding protein. The stiffness of SUMO1 (measured as a spring constant for the deformation response along the line joining the N- and C-termini) increased upon SBM binding from ∼1 N/m to ∼3.5 N/m. The relatively higher flexibility of ligand-free SUMO1 might play a role in accessing various conformations before binding to a target.

Single-Molecule Force Spectroscopy on Unfolded and Intrinsically Disordered Proteins

Transcription factors and cell-signaling proteins contain a hyper abundance of domains or segments that are intrinsically disordered (ID) under native conditions. In many cases, intrinsically disordered proteins (IDPs) or ID segments undergo coupled folding and binding and this transition is associated to its biological role. Many techniques for dissecting folding are not readily amenable to IDPs because they are aggregation prone and insoluble. Single-molecule methods provide an avenue to mitigate these issues. We have performed single-molecule force spectroscopy experiments on a Trimethylamine N-oxide (TMAO) refolded IDP and found that this IDP unfolds cooperatively under mechanical force. The data are rich in information and reveal that what appears to be a two-state transition in bulk is actually multi-state at the single-molecule level. We found that the analysis methods typically employed in such experiments may not be applicable because of the high concentrations of TMAO. To address these issues, we have designed unfolded mutants of T4 lysozyme using a random mutagenesis phenotypic screen. We are actively performing both bulk and single-molecule experiments to characterize unfolded mutants of T4 Lysozyme and TMAO refolded IDPs.