Single-molecule Force-clamp Spectroscopy Research Articles

Forcing the reversibility of a mechanochemical reaction

Mechanical force modifies the free-energy surface of chemical reactions, often enabling thermodynamically unfavoured reaction pathways. Most of our molecular understanding of force-induced reactivity is restricted to the irreversible homolytic scission of covalent bonds and ring-opening in polymer mechanophores. Whether mechanical force can by-pass thermodynamically locked reactivity in heterolytic bimolecular reactions and how this impacts the reaction reversibility remains poorly understood. Using single-molecule force-clamp spectroscopy, here we show that mechanical force promotes the thermodynamically disfavored SN2 cleavage of an individual protein disulfide bond by poor nucleophilic organic thiols. Upon force removal, the transition from the resulting high-energy unstable mixed disulfide product back to the initial, low-energy disulfide bond reactant becomes suddenly spontaneous, rendering the reaction fully reversible. By rationally varying the nucleophilicity of a series of small thiols, we demonstrate how force-regulated chemical kinetics can be finely coupled with thermodynamics to predict and modulate the reversibility of bimolecular mechanochemical reactions.

Tailoring protein nanomechanics with chemical reactivity

The nanomechanical properties of elastomeric proteins determine the elasticity of a variety of tissues. A widespread natural tactic to regulate protein extensibility lies in the presence of covalent disulfide bonds, which significantly enhance protein stiffness. The prevalent in vivo strategy to form disulfide bonds requires the presence of dedicated enzymes. Here we propose an alternative chemical route to promote non-enzymatic oxidative protein folding via disulfide isomerization based on naturally occurring small molecules. Using single-molecule force-clamp spectroscopy, supported by DFT calculations and mass spectrometry measurements, we demonstrate that subtle changes in the chemical structure of a transient mixed-disulfide intermediate adduct between a protein cysteine and an attacking low molecular-weight thiol have a dramatic effect on the protein’s mechanical stability. This approach provides a general tool to rationalize the dynamics of S-thiolation and its role in modulating protein nanomechanics, offering molecular insights on how chemical reactivity regulates protein elasticity.

Protein S-sulfenylation is a fleeting molecular switch that regulates non-enzymatic oxidative folding.

The post-translational modification S-sulfenylation functions as a key sensor of oxidative stress. Yet the dynamics of sulfenic acid in proteins remains largely elusive due to its fleeting nature. Here we use single-molecule force-clamp spectroscopy and mass spectrometry to directly capture the reactivity of an individual sulfenic acid embedded within the core of a single Ig domain of the titin protein. Our results demonstrate that sulfenic acid is a crucial short-lived intermediate that dictates the protein's fate in a conformation-dependent manner. When exposed to the solution, sulfenic acid rapidly undergoes further chemical modification, leading to irreversible protein misfolding; when cryptic in the protein's microenvironment, it readily condenses with a neighbouring thiol to create a protective disulfide bond, which assists the functional folding of the protein. This mechanism for non-enzymatic oxidative folding provides a plausible explanation for redox-modulated stiffness of proteins that are physiologically exposed to mechanical forces, such as cardiac titin.

Linking Mechanochemistry to Protein Folding at the Single Bond Level

The impact of post translational modifications (PTM) on protein folding remains elusive. S-sulfenylation is the first oxidation step of cysteinyl thiols to create sulfenic acid. This PTM has emerged as a key sensor that modulates protein function and redox signalling. Here we use single molecule force clamp spectroscopy to create a stable sulfenic acid conformation by promoting an OH- nucleophilic attack on a single disulfide bond embedded within the core of the 27th immunoglobulin domain of titin (I27). By applying a tailor-designed force protocol, we set out to study the effect of S-sulfenylation on the oxidative folding of the I27 protein. We observe that sulfenic acid is a necessary precursor that facilitates enzymatic-free disulfide bond reformation. Furthermore, this disulfide reformation mechanism competes with the evolution of sulfenic acid to higher oxidation state species, which ultimately triggers protein misfolding. These results demonstrate that the oxidation state of a single amino acid within a protein has the power to completely alter the outcome of protein folding.

Chaperone-Mediated Mechanical Protein Folding at the Single Molecule Level

DnaK, DnaJ and GrpE heat shock proteins form the main conserved chaperone system in Escherichia coli. Such a complex chaperone system participates in the correct folding of emergent polypeptides in the ribosome to ensure that they reach their native structure, while preventing the aggregation of proteins under stress conditions in the cell. It is well known from bulk experiments that DnaK and DnaJ accomplish these functions by binding to both unfolded and partially unfolded protein substrates. However, the extended protein conformations that characterise the nascent proteins in the ribosome are rarely accessible by biochemistry denaturing experiments. Moreover, the detailed conformation of the substrates required for an optimal chaperone binding still remains controversial. Here we use single molecule force-clamp spectroscopy AFM to study the effect of molecular chaperones on the mechanical folding of individual proteins. This approach enables to monitor the individual folding trajectories from highly extended states, and to dissect the different conformational ensembles visited by the single protein during its folding pathway. Our experiments reveal that DnaJ binds to the unfolded state of Ubiquitin (which contains the consensus binding sequence GKQLEDG) with more affinity than to the molten globule, collapsed state. DnaJ binding blocks ubiquitin refolding by decreasing its folding efficiency by over 40%, thus highlighting its “holdase” functionality. By contrast, DnaK presents instead a higher affinity for the molten globule conformations. Crucially, both proteins, together with GrpE, readily increase the refolding of Ubiquitin by over 10%, within an ATP-mediated process. Altogether, our nanomechanical experiments demonstrate that the binding of different chaperones to a unique substrate is highly conformation-dependent, enabling us to postulate a kinetic scheme that underlies the synergetic functionality of a complex chaperone system, at the single molecule level.

A Smoluchowski Equation for Force-Modulated Chemistry in Single Molecule Pulling Experiments

Thioredoxins (Trx) are a class of enzymes, which catalyze the reduction of disulphide bonds between two cysteine residues, commonly found in proteins. An experimental investigation into the reaction mechanisms employed by various species of Trx was carried out by Perez-Jimenez et al. using single molecule force-clamp spectroscopy. The experiment involved applying a pulling force along the disulfide bond of the protein substrate, and measuring the rate of the Trx-catalyzed reduction as a function of the pulling force. One interesting finding of the experiment was that some forms of thioredoxin exhibit a biphasic relationship for reduction rate as a function of force magnitude. For this project, a mathematical model of this system was created, which employs a Smoluchowski formalism in the vein of Agmon-Hopfied or Sumi-Markus models. The model describes the time evolution of the probability distribution function of the protein's configuration within a space defined as the internal protein coordinate, as it diffuses over a potential which distorts under the applied force, while losing probability density to the Bell model type “sink” term, which is also a function of the applied force, representing reactants going to products (disulphide bond cleavage). By numerically solving the Smoluchowski equation and integrating the resulting surface over both time and the protein coordinate to calculate lifetime for increasing values of applied force, the model successfully reproduced the experimentally observed values for disulphide bond reduction rate as a function of applied force. Parameterizing the Smoluchowski equation to fit the experimentally measured data points provided a means of drawing insights into a physical interpretation of the model including the relationship between degree of biphasic behavior and the distance along the reaction coordinate from the bottom of the reactant well to the top of the transition state.

Resolving the molecular mechanism of cadherin catch bond formation.

Classical cadherin Ca(2+)-dependent cell-cell adhesion proteins play key roles in embryogenesis and in maintaining tissue integrity. Cadherins mediate robust adhesion by binding in multiple conformations. One of these adhesive states, called an X-dimer, forms catch bonds that strengthen and become longer lived in the presence of mechanical force. Here we use single-molecule force-clamp spectroscopy with an atomic force microscope along with molecular dynamics and steered molecular dynamics simulations to resolve the molecular mechanisms underlying catch bond formation and the role of Ca(2+) ions in this process. Our data suggest that tensile force bends the cadherin extracellular region such that they form long-lived, force-induced hydrogen bonds that lock X-dimers into tighter contact. When Ca(2+) concentration is decreased, fewer de novo hydrogen bonds are formed and catch bond formation is eliminated.

Resolving the Molecular Determinants of Cadherin Catch Bond Formation

Classical cadherin Ca2+-dependent cell-cell adhesion proteins play key roles in embryogenesis and in maintaining tissue integrity. Cadherins mediate robust adhesion by binding in multiple conformations. We recently showed that one of these conformations, called an X-dimer, forms catch bonds that strengthen and become longer lived in the presence of mechanical stress. Here we use single molecule force clamp spectroscopy with an Atomic Force Microscope along with Molecular Dynamics and Steered Molecular Dynamics simulations to identify key interactions that mediate X-dimer catch bond formation and to resolve the role of Ca2+ ions in this process. We show that tensile force bends the cadherin extracellular region such that they form force-induced hydrogen bonds that lock X-dimers into tighter contact. When Ca2+ concentration is decreased, these hydrogen bonds are eliminated and catch bond formation is abolished. Based on these results, we formulate a simple ‘flex and lock’ kinetic scheme that quantitatively describes X-dimer catch bonds.

Large-Scale Modulation of Titin Elasticity by S-Glutathionylation of Cryptic Cysteines

The giant elastic protein titin is a determinant factor in how much blood fills the left ventricle during diastole, and thus in the etiology of heart disease. Titin has been identified as a target of S-glutathionylation, an end product of the nitric oxide signaling cascade that increases cardiac muscle elasticity. However, it is unknown whether S-glutathionylation regulates the elasticity of titin and cardiac tissue. Here, we use homology modeling techniques to show that most immunoglobulin (Ig) domains in the elastic I-band of titin contain cryptic cysteines, which are potential targets of S-glutathionylation triggered by physiological mechanical protein unfolding in the heart. We choose I91 as a representative Ig domain of titin to investigate the effects of S-glutathionylation in the elasticity of the protein. Using single-molecule force-clamp spectroscopy, we demonstrate that mechanical unfolding of I91 exposes two buried cysteine residues, which then can be S-glutathionylated by oxidized glutathione in the solution. S-glutathionylation of cryptic cysteines greatly decreases the mechanical stability of I91, which unfolds at a rate two orders of magnitude faster following S-glutathionylation. In addition, S-glutathionylation severely compromises the ability of I91 to fold. Both effects, which are fully reversed by the enzyme glutaredoxin, soften the I91 domain. When extrapolated to all the Ig domains in the I-band, our observations predict that S-glutathionylation can trigger a highly extensible state of titin. Indeed, we show that S-glutathionylation of cryptic cysteines in titin mediates mechano-chemical modulation of the elasticity of human cardiomyocytes. Monte Carlo simulations illustrate that large-scale regulation of the elasticity of titin through posttranslational modification of cryptic cysteines can be achieved on time scales of minutes to hours. We propose that posttranslational modification of cryptic residues is a general mechanism to regulate tissue elasticity.

Posttranslational Modifications as Modulators of Mechanical Protein Folding

Understanding the molecular basis of muscle elasticity is an important challenge in medicine. For example, mutations in the giant elastic protein titin are implicated in the etiology of dilated cardiomyopathies. We use single molecule force-clamp spectroscopy to study the folding and unfolding dynamics of single polyproteins composed of tandem repeats of titin Ig domains. Protein elasticity is due to the diffusional extension and collapse of a polyprotein along its free energy landscape. Using single molecule force-clamp traces we calculate the free energy of a polyprotein along its stretching coordinate under different mechanical force regimes. At high stretching forces the free energy is dominated by changes in entropy as the molecule unfolds and extends. When the force is quenched, enthalpic interactions become important as the polyprotein slowly progresses from collapsed states to molten globules and then to the mechanically stable native state. We also show that while the polyprotein is in the extended state, cysteine residues that are normally buried become exposed to the solution where they can be post-translationally modified. We have found that S-glutathionylation of such cryptic cysteines greatly decreases the mechanical stability of an Ig domain as well as its ability to fold, favoring more extensible states in titin. Thus, the free energy landscape of a protein extending and folding under force is strongly modulated by the redox state of a cell. We propose that posttranslational modification of cryptic cysteine residues is a major regulatory pathway of tissue elasticity. Accurate predictions of elastic phenotypes in titin will be possible after we fully understand how cellular chemistry modulates the physics of a polypeptide extending under force.

Force dependency of biochemical reactions measured by single-molecule force-clamp spectroscopy

Here we describe a protocol for using force-clamp spectroscopy to precisely quantify the effect of force on biochemical reactions. A calibrated force is used to control the exposure of reactive sites in a single polyprotein substrate composed of repeated domains. The use of polyproteins allows the identification of successful single-molecule recordings from unambiguous mechanical unfolding fingerprints. Biochemical reactions are then measured directly by detecting the length changes of the substrate held at a constant force. We present the layout of a force-clamp spectrometer along with protocols to design and conduct experiments. These experiments measure reaction kinetics as a function of applied force. We show sample data of the force dependency of two different reactions, protein unfolding and disulfide reduction. These data, which can be acquired in just a few days, reveal mechanistic details of the reactions that currently cannot be resolved by any other technique.

Ultrafast force-clamp spectroscopy of single molecules reveals load dependence of myosin working stroke

We describe a dual-trap force-clamp configuration that applies constant loads between a binding protein and an intermittently interacting biological polymer. The method has a measurement delay of only ∼10 μs, allows detection of interactions as brief as ∼100 μs and probes sub-nanometer conformational changes with a time resolution of tens of microseconds. We tested our method on molecular motors and DNA-binding proteins. We could apply constant loads to a single motor domain of myosin before its working stroke was initiated (0.2-1 ms), thus directly measuring its load dependence. We found that, depending on the applied load, myosin weakly interacted (<1 ms) with actin without production of movement, fully developed its working stroke or prematurely detached (<5 ms), thus reducing the working stroke size with load. Our technique extends single-molecule force-clamp spectroscopy and opens new avenues for investigating the effects of forces on biological processes.

Towards a General Platform to Study Single-Bond Chemistry Under Force

The application of force to reagents that participate in a chemical reaction can probe the transition state of the reaction with sub-Angstrom resolution. Using single-molecule force-clamp spectroscopy, this approach has been extensively applied to the cleavage of disulfide bonds in proteins. However, to date there is no methodology that can expand this class of experiments to bonds not naturally present in proteins. Here, we introduce an experimental platform with the potential to fulfill the requirements to perform single-bond rupture determinations on any covalent bond.

A Conditional Gating Mechanism Assures the Integrity of the Molecular Force-Sensor Titin Kinase

As more and more recent investigations point out, force plays an important role in cellular regulation mechanisms. Biological responses to mechanical stress are often based on force-induced conformational changes of single molecules. The force sensor, titin kinase, is involved in a signaling complex that regulates protein turnover and transcriptional adaptation in striated muscle. The structural architecture of such a force sensor determines its response to force and must assure both activity and mechanical integrity, which are prerequisites for its function. Here, we use single-molecule force-clamp spectroscopy to show that titin kinase is organized in such a way that the regulatory domains have to unfold before secondary structure elements that determine the overall fold and catalytic function. The stepwise unfolding over many barriers with a topologically determined sequence assures that the protein can react to force by conformational changes while maintaining its structural integrity.

Direct Quantification of the Attempt Frequency Determining the Mechanical Unfolding of Ubiquitin Protein

Understanding protein dynamics requires a comprehensive knowledge of the underlying potential energy surface that governs the motion of each individual protein molecule. Single molecule mechanical studies have provided the unprecedented opportunity to study the individual unfolding pathways along a well defined coordinate, the end-to-end length of the protein. In these experiments, unfolding requires surmounting an energy barrier that separates the native from the extended state. The calculation of the absolute value of the barrier height has traditionally relied on the assumption of an attempt frequency, υ(‡). Here we used single molecule force-clamp spectroscopy to directly determine the value of υ(‡) for mechanical unfolding by measuring the unfolding rate of the small protein ubiquitin at varying temperatures. Our experiments demonstrate a significant effect of the temperature on the mechanical rate of unfolding. By extrapolating the unfolding rate in the absence of force for different temperatures, varying within the range spanning from 5 to 45 °C, we measured a value for the activation barrier of ΔG(‡) = 71 ± 5 kJ/mol and an exponential prefactor υ(‡) ∼4 × 10(9) s(-1). Although the measured prefactor value is 3 orders of magnitude smaller than the value predicted by the transition state theory (∼6 × 10(12) s(-1)), it is 400-fold higher than that encountered in analogous experiments studying the effect of temperature on the reactivity of a protein-embedded disulfide bond (∼10(7) M(-1) s(-1)). This approach will allow quantitative characterization of the complete energy landscape of a folding polypeptide from highly extended states, of capital importance for proteins with elastic function.

Kinetic measurements on single-molecule disulfide bond cleavage.

We use single-molecule force clamp spectroscopy (SMFCS) to explore the reactivity of tris(2-carboxyethyl)phosphine (TCEP), 1, 4-dl-dithiothreitol (DTT) and hydrosulfide anion (HS(-)) on disulfide bonds within a mechanically stretched polypeptide. The single-bond level bimolecular nucleophilic substitution (S(N)2) events are recorded at a series of precisely controlled temperatures so that the Arrhenius kinetic parameters, that is, the height of the activation energy barrier (E(a)) and the attempting frequency (A) of the chemical reactions, can be determined. The values of A are typically at the order of 10(7) M(-1) s(-1), which is far lower than that predicted by the transition-state theory, in which A is given by k(B)T/h and around 10(12) M(-1) s(-1) at room temperature. Furthermore, E(a) is derived to be 30-40 kJ/mol, which can be lowered by ∼6-8% with every 100 pN mechanical force applied. The correlation of the A and E(a) with the molecular structures reveals that the relative magnitude of these two parameters cannot be simply judged from the size of the molecule or the nucleophilicity of the attacking atom. The comparison of the influences on the reaction rate induced by force and temperature indicates an equivalent accelerating effect by every 50 pN or 10 K increment, giving for the first time the relationship between mechanical and thermal effects on a single-molecule S(N)2 chemical reaction.

Protein Unfolding and Chemical Reactions Under Force: Complexity Versus Simplicity

Elucidating the dynamics of reactant molecules along the particular pathway that connects them with the resulting products is a cornerstone of modern chemistry. In general, for small systems composed of only a few molecules, the valley connecting reactants with products is narrow and the position of the transition state is highly localized. By contrast, in the case of more complex reactions involving bigger molecular systems such as proteins, the potential energy surfaces become rougher, which typically results in heterogeneous reaction pathways with multiple transition state structures. In both cases, detailed information on the energy landscapes has been mainly restricted to the results obtained using computational methods. Single molecule force-clamp spectroscopy can be now used to directly monitor the individual unfolding pathways of single proteins and the reactive trajectories corresponding to the chemical reduction of a single disulfide bond embedded within the core of a protein. Herein, we compare the effect of a mechanical constant force on the energy landscape of two different reactions; the unfolding of a single protein and the reduction of a single disulfide bond, both occurring within the same single protein. Our results demonstrate that the native state of the 27th immunoglobulin-like domain of titin is rough and probably composed of a set of similar structures with slightly different energy. Interestingly, shortening of the I27 protein by the presence of disulfide bonds has a strong effect on the measured heterogeneity in the unfolding pathways. Our results are quantitatively discussed within the framework of the static disorder theory. In sharp contrast with these results, the energy landscape of an SN2 chemical reaction, occurring within a much shorter length-scale, is smooth, implying a high degree of homogeneity within the individual reactive pathways connecting the reactants with the reaction products.

Single-Molecule Force-Clamp Spectroscopy: Dwell Time Analysis and Practical Considerations

Single-molecule force-clamp spectroscopy has become a powerful tool for studying protein folding/unfolding, bond rupture, and enzymatic reactions. Different methods have been developed to analyze force-clamp spectroscopy data on polyproteins to obtain kinetic parameters characterizing the mechanical unfolding of proteins, which are often modeled as a two-state process (a Poisson process). However, because of the finite number of domains in polyproteins, the statistical analysis of the force-clamp spectroscopy data is different from that of a classical Poisson process, and the equivalency of different analysis methods remains to be proven. In this article, we show that these methods are equivalent and lead to accurate measurements of the unfolding rate constant. We also demonstrate that distinct from the constant-pulling-velocity experiments, in which the unfolding rate extracted from the data is dependent on the number of protein domains in the polyproteins (the N effect), force-clamp experiments do not show any N effect. Using a simulated data set, we also highlighted important practical considerations that one needs to take into account when using the single-molecule force-clamp spectroscopy technique to characterize the unfolding energy landscape of proteins.

Paleoenzymology at the Single-Molecule Level: Probing the Chemistry of Resurrected Enzymes with Force-Clamp Spectroscopy

A journey back in time is possible at the molecular level by resurrecting proteins from extinct organisms. In the last two decades several methods based on statistical theory have been developed to computationally reconstruct ancestral protein sequences. Laboratory resurrection of these ancestral proteins provides an excellent opportunity to explore aspects of ancient life that cannot be inferred from fossil records. Here we report the resurrection of ancestral thioredoxin enzymes (Trx) from the Precambrian era, dating back between 1.5 to 4 billion years (Gyr). Using single molecule force-clamp spectroscopy we demonstrate that all ancestral enzymes efficiently reduce disulfide bonds. From the force-dependency of the rate of reduction of an engineered substrate, we conclude that the Precambrian enzymes have similar chemical mechanisms of reduction that the extant enzymes. By contrast, the resurrected enzymes show thermal stabilities 20 to 30 °C higher than those of modern E. coli and human Trx as revealed by Differential Scanning Calorimetry (DSC). This high thermostability illustrate that ancient organisms lived in hot environments that have progressively cooled. Trx enzymes adapted to these environmental temperatures with similar chemical mechanisms than those observed in extant Trxs. Our work demonstrates that the combination of single molecule force spectroscopy together with the resurrection of ancestral proteins is a powerful new approach to study molecular evolution and the sequence-chemistry relationship in enzymes.

Diversity of chemical mechanisms in thioredoxin catalysis revealed by single-molecule force spectroscopy

Thioredoxins are oxido-reductase enzymes present in all organisms, catalyzing the reduction of disulfide bonds in proteins. By applying a calibrated force to a substrate disulfide, the chemical mechanisms of Trx catalysis can be examined in detail at the single molecule level. Here we use single molecule force-clamp spectroscopy to explore the chemical evolution of Trx catalysis by probing the chemistry of eight different thioredoxin enzymes. While all Trxs show a characteristic Michaelis-Menten mechanism detected when the disulfide bond is stretched at low forces, two different chemical behaviors distinguish bacterial from eukaryotic-origin Trxs at high forces. Eukaryotic-origin Trxs reduce disulfide bonds through a single-electron transfer reaction (SET) whereas bacterial-origin Trxs exhibit both nucleophilic substitution (SN2) and SET reactions. A computational analysis of Trx structures identifies the evolution of the binding groove as an important factor controlling the chemistry of Trx catalysis.