S-glutathionylation of cryptic cysteines enhances titin elasticity by blocking protein folding.

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 how S-glutathionylation may regulate the elasticity of titin and cardiac tissue. Here, we show that mechanical unfolding of titin immunoglobulin (Ig) domains exposes buried cysteine residues, which then can be S-glutathionylated. S-glutathionylation of cryptic cysteines greatly decreases the mechanical stability of the parent Ig domain as well as its ability to fold. Both effects favor a more extensible state of titin. Furthermore, we demonstrate that S-glutathionylation of cryptic cysteines in titin mediates mechanochemical modulation of the elasticity of human cardiomyocytes. We propose that posttranslational modification of cryptic residues is a general mechanism to regulate tissue elasticity.

Protein folding drives disulfide formation.

PDI catalyzes the oxidative folding of disulfide-containing proteins. However, the sequence of reactions leading to a natively folded and oxidized protein remains unknown. Here we demonstrate a technique that enables independent measurements of disulfide formation and protein folding. We find that non-native disulfides are formed early in the folding pathway and can trigger misfolding. In contrast, a PDI domain favors native disulfides by catalyzing oxidation at a late stage of folding. We propose a model for cotranslational oxidative folding wherein PDI acts as a placeholder that is relieved by the pairing of cysteines caused by substrate folding. This general mechanism can explain how PDI catalyzes oxidative folding in a variety of structurally unrelated substrates.

Protease power strokes force proteins to unfold.

ATP-dependent proteases degrade proteins in the cytosol of cells. Two recent articles, by Aubin-Tam et al. (2011) and Maillard et al. (2011 [this issue]), use single-molecule optical tweezers to show directly that these molecular machines use the energy derived from ATP hydrolysis to mechanically unfold and translocate its substrates into the proteolytic chamber.

Identical sequences, different behaviors: Protein diversity captured at the single-molecule level.

The classical "one sequence, one structure, one function" paradigm has shaped much of our intuition of how proteins work inside the cell. Partially due to the insight provided by bulk biochemical assays, individual biomolecules are often assumed to behave as identical entities, and their characterization relies on ensemble averages that flatten any conformational diversity into a unique phenotype. While the emergence of single-molecule techniques opened the gates to interrogating individual molecules, technical shortcomings typically limit the duration of these measurements, which precludes a complete characterization of an individual protein and, hence, capturing the heterogeneity among molecular populations. Here, we introduce an ultrastable magnetic tweezers design, which enables us to measure the folding dynamics of a single protein during several uninterrupted days with high temporal and spatial resolution. Thanks to this instrumental development, we fully characterize the nanomechanics of two proteins with a very distinct force response, the talin R3IVVI domain and protein L. Days-long recordings on the same protein individual accumulate thousands of folding transitions with submicrosecond resolution, allowing us to reconstruct their free energy landscapes and describe how they evolve with force. By mapping the nanomechanical identity of many different protein individuals, we directly capture their molecular diversity as a quantifiable dispersion on their force response and folding kinetics. By significantly expanding the measurable timescales, our instrumental development offers a tool for profiling individual molecules, opening the gates to directly characterizing biomolecular heterogeneity.

Talin folding as the tuning fork of cellular mechanotransduction.

Cells continually sample their mechanical environment using exquisite force sensors such as talin, whose folding status triggers mechanotransduction pathways by recruiting binding partners. Mechanical signals in biology change quickly over time and are often embedded in noise; however, the mechanics of force-sensing proteins have only been tested using simple force protocols, such as constant or ramped forces. Here, using our magnetic tape head tweezers design, we measure the folding dynamics of single talin proteins in response to external mechanical noise and cyclic force perturbations. Our experiments demonstrate that talin filters out external mechanical noise but detects periodic force signals over a finely tuned frequency range. Hence, talin operates as a mechanical band-pass filter, able to read and interpret frequency-dependent mechanical information through its folding dynamics. We describe our observations in the context of stochastic resonance, which we propose as a mechanism by which mechanosensing proteins could respond accurately to force signals in the naturally noisy biological environment.

Ephemeral states in protein folding under force captured with a magnetic tweezers design.

Magnetic tape heads are ubiquitously used to read and record on magnetic tapes in technologies as diverse as old VHS tapes, modern hard-drive disks, or magnetic bands on credit cards. Their design highlights the ability to convert electric signals into fluctuations of the magnetic field at very high frequencies, which is essential for the high-density storage demanded nowadays. Here, we twist this conventional use of tape heads to implement one in a magnetic tweezers design, which offers the unique capability of changing the force with a bandwidth of ∼10 kHz. We calibrate our instrument by developing an analytical expression that predicts the magnetic force acting on a superparamagnetic bead based on the Karlqvist approximation of the magnetic field created by a tape head. This theory is validated by measuring the force dependence of protein L unfolding/folding step sizes and the folding properties of the R3 talin domain. We demonstrate the potential of our instrument by carrying out millisecond-long quenches to capture the formation of the ephemeral molten globule state in protein L, which has never been observed before. Our instrument provides the capability of interrogating individual molecules under fast-changing forces with a control and resolution below a fraction of a piconewton, opening a range of force spectroscopy protocols to study protein dynamics under force.

The Work of Titin Protein Folding as a Major Driver in Muscle Contraction.

Single-molecule atomic force microscopy and magnetic tweezers experiments have demonstrated that titin immunoglobulin (Ig) domains are capable of folding against a pulling force, generating mechanical work that exceeds that produced by a myosin motor. We hypothesize that upon muscle activation, formation of actomyosin cross bridges reduces the force on titin, causing entropic recoil of the titin polymer and triggering the folding of the titin Ig domains. In the physiological force range of 4-15 pN under which titin operates in muscle, the folding contraction of a single Ig domain can generate 200% of the work of entropic recoil and occurs at forces that exceed the maximum stalling force of single myosin motors. Thus, titin operates like a mechanical battery, storing elastic energy efficiently by unfolding Ig domains and delivering the charge back by folding when the motors are activated during a contraction. We advance the hypothesis that titin folding and myosin activation act as inextricable partners during muscle contraction.

Molecular strategy for blocking isopeptide bond formation in nascent pilin proteins.

Bacteria anchor to their host cells through their adhesive pili, which must resist the large mechanical stresses induced by the host as it attempts to dislodge the pathogens. The pili of gram-positive bacteria are constructed as a single polypeptide made of hundreds of pilin repeats, which contain intramolecular isopeptide bonds strategically located in the structure to prevent their unfolding under force, protecting the pilus from degradation by extant proteases and oxygen radicals. Here, we demonstrate the design of a short peptide that blocks the formation of the isopeptide bond present in the pilin Spy0128 from the human pathogen Streptococcus pyogenes, resulting in mechanically labile pilin domains. We use a combination of protein engineering and atomic-force microscopy force spectroscopy to demonstrate that the peptide blocks the formation of the native isopeptide bond and compromises the mechanics of the domain. While an intact Spy0128 is inextensible at any force, peptide-modified Spy0128 pilins readily unfold at very low forces, marking the abrogation of the intramolecular isopeptide bond as well as the absence of a stable pilin fold. We propose that isopeptide-blocking peptides could be further developed as a type of highly specific antiadhesive antibiotics to treat gram-positive pathogens.

CnaA domains in bacterial pili are efficient dissipaters of large mechanical shocks.

Pathogenic bacteria adhere despite severe mechanical perturbations induced by the host, such as coughing. In Gram-positive bacteria, extracellular protein appendages termed pili are necessary for adherence under mechanical stress. However, little is known about the behavior of Gram-positive pili under force. Here, we demonstrate a mechanism by which Gram-positive pili are able to dissipate mechanical energy through mechanical unfolding and refolding of isopeptide bond-delimited polypeptide loops present in Ig-type CnaA domains. Using single-molecule force spectroscopy, we find that these loops of the pilus subunit SpaA of the SpaA-type pilus from Corynebacterium diphtheriae and FimA of the type 2 pilus from Actinomyces oris unfold and extend at forces that are the highest yet reported for globular proteins. Loop refolding is limited by the hydrophobic collapse of the polypeptide and occurs in milliseconds. Remarkably, both SpaA and FimA initially refold to mechanically weaker intermediates that recover strength with time or ligand binding. Based on the high force extensibility, CnaA-containing pili can dissipate ∼28-fold as much energy compared with their inextensible counterparts before reaching forces sufficient to cleave covalent bonds. We propose that efficient mechanical energy dissipation is key for sustained bacterial attachment against mechanical perturbations.

Mechanical forces regulate the reactivity of a thioester bond in a bacterial adhesin.

Bacteria must withstand large mechanical shear forces when adhering to and colonizing hosts. Recent structural studies on a class of Gram-positive bacterial adhesins have revealed an intramolecular Cys-Gln thioester bond that can react with surface-associated ligands to covalently anchor to host surfaces. Two other examples of such internal thioester bonds occur in certain anti-proteases and in the immune complement system, both of which react with the ligand only after the thioester bond is exposed by a proteolytic cleavage. We hypothesized that mechanical forces in bacterial adhesion could regulate thioester reactivity to ligand analogously to such proteolytic gating. Studying the pilus tip adhesin Spy0125 of Streptococcus pyogenes, we developed a single molecule assay to unambiguously resolve the state of the thioester bond. We found that when Spy0125 was in a folded state, its thioester bond could be cleaved with the small-molecule nucleophiles methylamine and histamine, but when Spy0125 was mechanically unfolded and subjected to forces of 50-350 piconewtons, thioester cleavage was no longer observed. For folded Spy0125 without mechanical force exposure, thioester cleavage was in equilibrium with spontaneous thioester reformation, which occurred with a half-life of several minutes. Functionally, this equilibrium reactivity allows thioester-containing adhesins to sample potential substrates without irreversible cleavage and inactivation. We propose that such reversible thioester reactivity would circumvent potential soluble inhibitors, such as histamine released at sites of inflammation, and allow the bacterial adhesin to selectively associate with surface-bound ligands.

Single-molecule Force Spectroscopy Predicts a Misfolded, Domain-swapped Conformation in human γD-Crystallin Protein.

Cataract is a protein misfolding disease where the size of the aggregate is directly related to the severity of the disorder. However, the molecular mechanisms that trigger the onset of aggregation remain unknown. Here we use a combination of protein engineering techniques and single-molecule force spectroscopy using atomic force microscopy to study the individual unfolding pathways of the human γD-crystallin, a multidomain protein that must remain correctly folded during the entire lifetime to guarantee lens transparency. When stretching individual polyproteins containing two neighboring HγD-crystallin monomers, we captured an anomalous misfolded conformation in which the ß1 and ß2 strands of the N terminus domain of two adjacent monomers swap. This experimentally elusive domain-swapped conformation is likely to be responsible for the increase in molecular aggregation that we measure in vitro. Our results demonstrate the power of force spectroscopy at capturing rare misfolded conformations with potential implications for the understanding of the molecular onset of protein aggregation.

Mechanical Deformation Accelerates Protein Ageing.

A hallmark of tissue ageing is the irreversible oxidative modification of its proteins. We show that single proteins, kept unfolded and extended by a mechanical force, undergo accelerated ageing in times scales of minutes to days. A protein forced to be continuously unfolded completely loses its ability to contract by folding, becoming a labile polymer. Ageing rates vary among different proteins, but in all cases they lose their mechanical integrity. Random oxidative modification of cryptic side chains exposed by mechanical unfolding can be slowed by the addition of antioxidants such as ascorbic acid, or accelerated by oxidants. By contrast, proteins kept in the folded state and probed over week-long experiments show greatly reduced rates of ageing. We demonstrate a novel approach whereby protein ageing can be greatly accelerated: the constant unfolding of a protein for hours to days is equivalent to decades of exposure to free radicals under physiological conditions.

Monitoring Oxidative Folding of a Single Protein Catalyzed by the Disulfide Oxidoreductase DsbA.

Oxidative folding, the process by which proteins fold and acquire disulfide bonds concurrently, is of critical importance for a wide range of biological processes. Generally, this process is catalyzed by oxidoreductase enzymes that facilitate oxidation and also bear chaperone functionality. Although this process has been well described qualitatively, fine yet important details remain obscured by a limited quantitative perspective, arising from the limitations in the application of bulk biochemical methods to the study of oxidative folding. In this work, we have applied single molecule force spectroscopy techniques to monitor in real time the process of oxidative folding as catalyzed by DsbA, the enzyme solely responsible for the catalysis of oxidative folding in the bacterial periplasm. We provide a quantitative and detailed description of the catalytic mechanism utilized by DsbA that offers insight into the entire sequence of events that occurs in the periplasm from the unfolded-reduced state to the folded-oxidized protein. We have compared our results with those of protein disulfide-isomerase, the eukaryotic counterpart of DsbA, allowing us to devise a general mechanism for oxidative folding that also reflects upon the physiological functions and demands of these enzymes in vivo.

A HaloTag Anchored Ruler for Week-Long Studies of Protein Dynamics.

Under physiological conditions, protein oxidation and misfolding occur with very low probability and on long times scales. Single-molecule techniques provide the ability to distinguish between properly folded and damaged proteins that are otherwise masked in ensemble measurements. However, at physiological conditions these rare events occur with a time constant of several hours, inaccessible to current single-molecule approaches. Here we present a magnetic-tweezers-based technique that allows, for the first time, the study of folding of single proteins during week-long experiments. This technique combines HaloTag anchoring, sub-micrometer positioning of magnets, and an active correction of the focal drift. Using this technique and protein L as a molecular template, we generate a magnet law by correlating the distance between the magnet and the measuring paramagnetic bead with unfolding/folding steps. We demonstrate that, using this magnet law, we can accurately measure the dynamics of proteins over a wide range of forces, with minimal dispersion from bead to bead. We also show that the force calibration remains invariant over week-long experiments applied to the same single proteins. The approach demonstrated in this Article opens new, exciting ways to examine proteins on the "human" time scale and establishes magnetic tweezers as a valuable technique to study low-probability events that occur during protein folding under force.

Elasticity, structure, and relaxation of extended proteins under force.

Force spectroscopies have emerged as a powerful and unprecedented tool to study and manipulate biomolecules directly at a molecular level. Usually, protein and DNA behavior under force is described within the framework of the worm-like chain (WLC) model for polymer elasticity. Although it has been surprisingly successful for the interpretation of experimental data, especially at high forces, the WLC model lacks structural and dynamical molecular details associated with protein relaxation under force that are key to the understanding of how force affects protein flexibility and reactivity. We use molecular dynamics simulations of ubiquitin to provide a deeper understanding of protein relaxation under force. We find that the WLC model successfully describes the simulations of ubiquitin, especially at higher forces, and we show how protein flexibility and persistence length, probed in the force regime of the experiments, are related to how specific classes of backbone dihedral angles respond to applied force. Although the WLC model is an average, backbone model, we show how the protein side chains affect the persistence length. Finally, we find that the diffusion coefficient of the protein's end-to-end distance is on the order of 10(8) nm(2)/s, is position and side-chain dependent, but is independent of the length and independent of the applied force, in contrast with other descriptions.

Altered thiol chemistry in human amyotrophic lateral sclerosis-linked mutants of superoxide dismutase 1.

Neurodegenerative diseases share a common characteristic, the presence of intracellular or extracellular deposits of protein aggregates in nervous tissues. Amyotrophic Lateral Sclerosis (ALS) is a severe and fatal neurodegenerative disorder, which affects preferentially motoneurons. Changes in the redox state of superoxide dismutase 1 (SOD1) are associated with the onset and development of familial forms of ALS. In human SOD1 (hSOD1), a conserved disulfide bond and two free cysteine residues can engage in anomalous thiol/disulfide exchange resulting in non-native disulfides, a hallmark of ALS that is related to protein misfolding and aggregation. Because of the many competing reaction pathways, traditional bulk techniques fall short at quantifying individual thiol/disulfide exchange reactions. Here, we adapt recently developed single-bond chemistry techniques to study individual disulfide isomerization reactions in hSOD1. Mechanical unfolding of hSOD1 leads to the formation of a polypeptide loop held by the disulfide. This loop behaves as a molecular jump rope that brings reactive Cys-111 close to the disulfide. Using force-clamp spectroscopy, we monitor nucleophilic attack of Cys-111 at either sulfur of the disulfide and determine the selectivity of the reaction. Disease-causing mutations G93A and A4V show greatly altered reactivity patterns, which may contribute to the progression of familial ALS.

Dynamics of equilibrium folding and unfolding transitions of titin immunoglobulin domain under constant forces.

The mechanical stability of force-bearing proteins is crucial for their functions. However, slow transition rates of complex protein domains have made it challenging to investigate their equilibrium force-dependent structural transitions. Using ultra stable magnetic tweezers, we report the first equilibrium single-molecule force manipulation study of the classic titin I27 immunoglobulin domain. We found that individual I27 in a tandem repeat unfold/fold independently. We obtained the force-dependent free energy difference between unfolded and folded I27 and determined the critical force (∼5.4 pN) at which unfolding and folding have equal probability. We also determined the force-dependent free energy landscape of unfolding/folding transitions based on measurement of the free energy cost of unfolding. In addition to providing insights into the force-dependent structural transitions of titin I27, our results suggest that the conformations of titin immunoglobulin domains can be significantly altered during low force, long duration muscle stretching.

The elastic free energy of a tandem modular protein under force.

Recent studies have provided a theoretical framework for including entropic elasticity in the free energy landscape of proteins under mechanical force. Accounting for entropic elasticity using polymer physics models has helped explain the hopping behavior seen in single molecule experiments in the low force regime. Here, we expand on the construction of the free energy of a single protein domain under force proposed by Berkovich et al. to provide a free energy landscape for N tandem domains along a continuous polypeptide. Calculation of the free energy of individual domains followed by their concatenation provides a continuous free energy landscape whose curvature is dominated by the worm-like chain at forces below 20 pN. We have validated our free energy model using Brownian dynamics and reproduce key features of protein folding. This free energy model can predict the effects of changes in the elastic properties of a multidomain protein as a consequence of biological modifications such as phosphorylation or the formation of disulfide bonds. This work lays the foundations for the modeling of tissue elasticity, which is largely determined by the properties of tandem polyproteins.

Rate limit of protein elastic response is tether dependent.

The elastic restoring force of tissues must be able to operate over the very wide range of loading rates experienced by living organisms. It is surprising that even the fastest events involving animal muscle tissues do not surpass a few hundred hertz. We propose that this limit is set in part by the elastic dynamics of tethered proteins extending and relaxing under a changing load. Here we study the elastic dynamics of tethered proteins using a fast force spectrometer with sub-millisecond time resolution, combined with Brownian and Molecular Dynamics simulations. We show that the act of tethering a polypeptide to an object, an inseparable part of protein elasticity in vivo and in experimental setups, greatly reduces the attempt frequency with which the protein samples its free energy. Indeed, our data shows that a tethered polypeptide can traverse its free-energy landscape with a surprisingly low effective diffusion coefficient D(eff) ~ 1,200 nm(2)/s. By contrast, our Molecular Dynamics simulations show that diffusion of an isolated protein under force occurs at D(eff) ~ 10(8) nm(2)/s. This discrepancy is attributed to the drag force caused by the tethering object. From the physiological time scales of tissue elasticity, we calculate that tethered elastic proteins equilibrate in vivo with D(eff) ~ 10(4)-10(6) nm(2)/s which is two to four orders magnitude smaller than the values measured for untethered proteins in bulk.

Conformational plasticity of the essential membrane-associated mannosyltransferase PimA from mycobacteria.

Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase (GT) that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannosides, lipomannan, and lipoarabinomannan, which are key glycolipids/lipoglycans of the mycobacterial cell envelope. PimA belongs to a large family of peripheral membrane-associated GTs for which the understanding of the molecular mechanism and conformational changes that govern substrate/membrane recognition and catalysis remains a major challenge. Here we used single molecule force spectroscopy techniques to study the mechanical and conformational properties of PimA. In our studies, we engineered a polyprotein containing PimA flanked by four copies of the well characterized I27 protein, which provides an unambiguous mechanical fingerprint. We found that PimA exhibits weak mechanical stability albeit displaying ß-sheet topology expected to unfold at much higher forces. Notably, PimA unfolds following heterogeneous multiple step mechanical unfolding pathways at low force akin to molten globule states. Interestingly, the ab initio low resolution envelopes obtained from small angle x-ray scattering of the unliganded PimA and the PimA·GDP complexed forms clearly demonstrate that not only the "open" and "closed" conformations of the GT-B enzyme are largely present in solution, but in addition, PimA experiences remarkable flexibility that undoubtedly corresponds to the N-terminal "Rossmann fold" domain, which has been proved to participate in protein-membrane interactions. Based on these results and on our previous experimental data, we propose a model wherein the conformational transitions are important for the mannosyltransferase to interact with the donor and acceptor substrates/membrane.