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Gram-positive bacteria use their adhesive pili to attach to host cells during early stages of a bacterial infection. These extracellular hair-like appendages experience mechanical stresses of hundreds of picoNewtons; however, the presence of an internal isopeptide bond prevents the pilus protein from unfolding. Here, we describe a method to interfere with nascent pili proteins through a peptide that mimics one of the β-strands of the molecule. By using AFM-based force spectroscopy, we study the isopeptide bond formation and the effect of the peptide in the elasticity of the pilus protein. This method could be used to afford a new strategy for mechanically targeted antibiotics by simply blocking the folding of the bacterial pilus protein.

Single-molecule methods using recombinant proteins have generated transformative hypotheses on how mechanical forces interact with titin in the sarcomere, enabling muscle contraction. However, testing these mechanical hypotheses on native titin in its natural environment has remained inaccessible to conventional genetics, biophysics and molecular biology tools. To overcome these limitations, here we demonstrate a genetically engineered knock-in mouse model carrying a HaloTag-TEV insertion in titin. Using our system, we have specifically severed the titin filament by digestion with TEV protease, and found that the response of muscle fibers to length changes requires mechanical transduction through titin’s intact polypeptide chain. HaloTag-based covalent tethering has enabled directed examination of the dynamics of native titin under physiological forces using recently developed magnetic tweezers. At physiological pulling forces lower than 10 pN, titin domains are readily recruited to the unfolded state, and produce 41.5 zJ mechanical work during refolding. Our results support an active role of titin in muscle contraction in coordination with actomyosin motors.

Single-molecule methods using recombinant proteins have generated transformative hypotheses on how mechanical forces are generated and sensed in biological tissues. However, testing these mechanical hypotheses on proteins in their natural environment remains inaccesible to conventional tools. To address this limitation, here we demonstrate a mouse model carrying a HaloTag-TEV insertion in the protein titin, the main determinant of myocyte stiffness. Using our system, we specifically sever titin by digestion with TEV protease, and find that the response of muscle fibers to length changes requires mechanical transduction through titin’s intact polypeptide chain. In addition, HaloTag-based covalent tethering enables examination of titin dynamics under force using magnetic tweezers. At pulling forces < 10 pN, titin domains are recruited to the unfolded state, and produce 41.5 zJ mechanical work during refolding. Insertion of the HaloTag-TEV cassette in mechanical proteins opens opportunities to explore the molecular basis of cellular force generation, mechanosensing and mechanotransduction., Testing mechanical forces on native molecules in natural environments remains a challenge. Here the authors engineer titin to carry a HaloTag-TEV insertion to allow analysis of dynamics under force in muscle fibers.

SummaryCurrent theories of muscle contraction propose that the power stroke of a myosin motor is the sole source of mechanical energy driving the sliding filaments of a contracting muscle. These models exclude titin, the largest protein in the human body, which determines the passive elasticity of muscles. Here, we show that stepwise unfolding/folding of titin immunoglobulin (Ig) domains occurs in the elastic I band region of intact myofibrils at physiological sarcomere lengths and forces of 6–8 pN. We use single-molecule techniques to demonstrate that unfolded titin Ig domains undergo a spontaneous stepwise folding contraction at forces below 10 pN, delivering up to 105 zJ of additional contractile energy, which is larger than the mechanical energy delivered by the power stroke of a myosin motor. Thus, it appears inescapable that folding of titin Ig domains is an important, but as yet unrecognized, contributor to the force generated by a contracting muscle.

Titin — the largest protein in the human body — spans half of the muscle sarcomere from the Z-disk to the M-band through a single polypeptide chain. More than 30 000 amino acid residues coded from a single gene (TTN, in humans Q8WZ42) form a long filamentous protein organized in individual globular domains concatenated in tandem. Owing to its location and close interaction with the other muscle filaments, titin is considered the third filament of muscle, after the thick-myosin and the thin-actin filaments.

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 AFM 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 novel type of highly-specific anti-adhesive antibiotics to treat Gram-positive pathogens.SignificanceAt the onset of an infection, Gram-positive bacteria adhere to host cells through their pili, filamentous structures built by hundreds of repeats of pilin proteins. These proteins can withstand large mechanical challenges without unfolding, remaining anchored to the host and resisting cleavage by proteases and oxygen radicals present in the targeted tissues. The key structural component that gives pilins mechanical resilience are internal isopeptide bonds, strategically placed so that pilins become inextensible structures. We target this bond by designing a blocking peptide that interferes with its formation during folding. We demonstrate that peptide-modified pilins lack mechanical stability and extend at low forces. We propose this strategy as a rational design of mechanical antibiotics, targeting the Achilles’ Heel of bacterial adhesion.

The activity of the ADP-dependent glucokinase from Thermococcus litoralis (TlGK) relies on the highly conserved motifs NXXE (i.e. Asn-Xaa-Xaa-Glu) and HXE (i.e. His-Xaa-Glu). Site-directed mutagenesis of residues Glu279 (HXE) and Glu308 (NXXE) leads to enzymes with highly reduced catalytic rates. The replacement of Glu308 by Gln increased the KM for MgADP(-) and was activated by free Mg(2+). On the other hand, HXE mutants did not affect the KM for MgADP(-), were still inhibited by free Mg(2+), and caused a large increase on KM for glucose and an 87-fold weaker binding of glucose onto the non-hydrolysable TlGK·AMP-AlF3 complex. Our findings put forward the fundamental role of the HXE motif in glucose binding during ternary complex formation.

Protein ageing may manifest as a mechanical disease that compromises tissue elasticity. As proved recently, while proteins respond to changes in force with an instantaneous elastic recoil followed by a folding contraction, aged proteins break bad, becoming unstructured polymers. Here, we explain this phenomenon in the context of a free energy model, predicting the changes in the folding landscape of proteins upon oxidative ageing. Our findings validate that protein folding under force is constituted by two separable components, polymer properties and hydrophobic collapse, and demonstrate that the latter becomes irreversibly blocked by oxidative damage. We run Brownian dynamics simulations on the landscape of protein L octamer, reproducing all experimental observables, for a naïve and damaged polyprotein. This work provides a unique tool to understand the evolving free energy landscape of elastic proteins upon physiological changes, opening new perspectives to predict age-related diseases in tissues.

A hallmark of tissue ageing is the irreversible oxidative modifications of its constituent 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, loses completely its ability to contract by folding becoming a labile polymer. Ageing rates vary amongst different types of 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 assay where 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

The active site of the Haloalkane Dehydrogenase (HaloTag) enzyme can be covalently attached to a chloroalkane ligand providing a mechanically strong tether, resistant to large pulling forces. Here we demonstrate the covalent tethering of protein L and I27 polyproteins between an atomic force microscopy (AFM) cantilever and a glass surface using HaloTag anchoring at one end and thiol chemistry at the other end. Covalent tethering is unambiguously confirmed by the observation of full length polyprotein unfolding, combined with high detachment forces that range up to ∼2000 pN. We use these covalently anchored polyproteins to study the remarkable mechanical properties of HaloTag proteins. We show that the force that triggers unfolding of the HaloTag protein exhibits a 4-fold increase, from 131 to 491 pN, when the direction of the applied force is changed from the C-terminus to the N-terminus. Force-clamp experiments reveal that unfolding of the HaloTag protein is twice as sensitive to pulling force compared to protein L and refolds at a slower rate. We show how these properties allow for the long-term observation of protein folding-unfolding cycles at high forces, without interference from the HaloTag tether.

Force-spectroscopy has been an essential tool for understanding how proteins fold and unfold, and their involvement in regulating cell responses to mechanical stimuli. Hence, it is of significance to establish an accurate free energy model of proteins under force capable of describing the mechanisms of protein folding. Even though numerous models have been proposed, the role of polymer physics and the changes in entropy involved in protein elasticity are still controversial. We have developed a model to construct the free energy of elastic proteins under force based on protein folding and polymer physics. The model is constructed by combining Morse and Gaussian potentials describing the enthalpic interactions in the folded state, and the freely jointed-chain (FJC) energy model accounting for the entropic changes during unfolding and stretching. The free parameters of each component are dependent on the protein and thus regulate its mechanical properties [J. Valle-Orero et al, BBRC 460 (2015)]. Here we aim to prove the accuracy of the model by reproducing force-spectroscopy data of a model protein, protein L (B1 domain from Peptostreptococcus magnus). We experimentally fit the force dependent extension of unfolded domains with the FJC using a contour length of 16.3 nm and a Kuhn length of 1.1 nm. The Morse depth of 2 kBT was established by measuring the probability that a domain folds at a given force. A Gaussian barrier of 10.5 kBT was chosen by comparing the experimental unfolding kinetics to those obtained using Brownian dynamics on our energy model. The excellent agreement of our model with experimental data validates and reinforces the theoretical foundations of our physics model. Furthermore, we demonstrate that changes in entropy during extending or collapsing a protein results in departure from simple Bell-like models.

The reaction catalyzed by E. coli Pfk-2 presents a dual-cation requirement. In addition to that chelated by the nucleotide substrate, an activating cation is required to obtain full activity of the enzyme. Only Mn 2+ and Mg 2+ can fulfill this role binding to the same activating site but the affinity for Mn 2+ is 13-fold higher compared to that of Mg 2+ . The role of the E190 residue, present in the highly conserved motif NXXE involved in Mg 2+ binding, is also evaluated in this behavior. The E190Q mutation drastically diminishes the kinetic affinity of this site for both cations. However, binding studies of free Mn 2+ and metal–Mant-ATP complex through EPR and FRET experiments between the ATP analog and Trp88, demonstrated that Mn 2+ as well as the metal–nucleotide complex bind with the same affinity to the wild type and E190Q mutant Pfk-2. These results suggest that this residue exert its role mainly kinetically, probably stabilizing the transition state and that the geometry of metal binding to E190 residue may be crucial to determine the catalytic competence.

Enzyme-substrate binding is a dynamic process intimately coupled to protein structural changes, which in turn changes the unfolding energy landscape. By the use of single-molecule force spectroscopy (SMFS), we characterize the open-to-closed conformational transition experienced by the hyperthermophilic adenine diphosphate (ADP)-dependent glucokinase from Thermococcus litoralis triggered by the sequential binding of substrates. In the absence of substrates, the mechanical unfolding of TlGK shows an intermediate 1, which is stabilized in the presence of Mg·ADP(-), the first substrate to bind to the enzyme. However, in the presence of this substrate, an additional unfolding event is observed, intermediate 1*. Finally, in the presence of both substrates, the unfolding force of intermediates 1 and 1* increases as a consequence of the domain closure. These results show that SMFS can be used as a powerful experimental tool to investigate binding mechanisms of different enzymes with more than one ligand, expanding the repertoire of protocols traditionally used in enzymology.

Advancements in single-molecule force spectroscopy techniques such as atomic force microscopy and magnetic tweezers allow investigation of how domain folding under force can play a physiological role. Combining these techniques with protein engineering and HaloTag covalent attachment, we investigate similarities and differences between four model proteins: I10 and I91-two immunoglobulin-like domains from the muscle protein titin, and two α + β fold proteins-ubiquitin and protein L. These proteins show a different mechanical response and have unique extensions under force. Remarkably, when normalized to their contour length, the size of the unfolding and refolding steps as a function of force reduces to a single master curve. This curve can be described using standard models of polymer elasticity, explaining the entropic nature of the measured steps. We further validate our measurements with a simple energy landscape model, which combines protein folding with polymer physics and accounts for the complex nature of tandem domains under force. This model can become a useful tool to help in deciphering the complexity of multidomain proteins operating under force.

The last 20 years have witnessed a revolution in our understanding of the mechanical behavior of proteins, driven largely by advances in single-molecule force spectroscopy (SMFS). However, direct single-molecule study of certain mechanical systems, such as muscle titin and the bacterial pilus, has remained out of reach due to their megadalton-scale sizes and due to difficulties of pulling at high forces. Here, we have implemented a strategy based on HaloTag and SpyCatcher-SpyTag chemistries that provides for modular hetero-polyprotein construction and fully covalent SMFS with Magnetic Tweezers (MT). We functionalized both the surface and probe with HaloTag chemistry to have end-to-end covalent attachments in MT, enabling hours-long recordings on single molecules held at high force (> 100 pN). In addition, we have incorporated the SpyCatcher-SpyTag protein conjugation system for macromolecule construction. Whereas SMFS has been limited to the study of octamers of up to ∼120 kDa, SpyCatcher-SpyTag chemistry extends the range to 2nd-order assemblies of polyproteins. We expressed polyproteins flanked with either SpyCatcher or SpyTag pairs, and assembled large heterogeneous molecules through sequential construction on MT surfaces or on magnetic beads. Applying MT-based SMFS, we observed folding/unfolding step sizes of the various constructs assembled into single hetero-polyproteins. To date, we have successfully assembled and recorded from a trimer of I27 octamers and from a hetero-trimer of LB1 and I27 polyproteins. These advances in covalent attachment and protein assembly set the stage for megadalton-scale construction of complex titin-like molecules and for their long-term study under force.

The elasticity of muscle is principally determined by the I-band region of titin, consisting of 40-100 immunoglobulin-like (Ig) domains and two unstructured segments. Traditional force spectroscopy of titin can study only eight tandem Ig domains at a time, and the dominant entropic effect of the massive native titin polypeptide is lost. Here, we have developed a knock-in mouse model to study titin in its entirety. We genetically encoded a HaloTag enzyme, flanked with two protease sites, in the constitutively expressed region of the I-band. This multi-tool mouse model permits: covalent protein immobilization for force spectroscopy, fluorescent labeling for sarcomere length measurements, and titin specific proteolysis for muscle mechanics experiments. The HaloTag, engineered close to the I-A junction of titin, allows titin immobilization on glass coverslips functionalized with chloroalkane chemistry. Using T12 antibody coated paramagnetic beads, the N-terminus of titin can be picked up and stretched with magnetic tweezers. For the first time, this has enabled the study of the mechanical properties of the entire intact titin I-band. For different muscle groups, the results show unfolding and refolding of individual Ig domains at forces in the physiological range, below 10 pN. Moreover, accumulated step size histograms show two populations in the unfolding and refolding transitions, suggesting the presence of reduced and oxidized disulfide bonds in several Ig domains. Additionally, fluorescent labeling of the HaloTag with Alexa488 conjugated chloroalkane permits accurate sarcomere length measurements of intact myofibrils. We propose muscle mechanics experiments in which stretched myofibrils are treated with TEV protease to cleave titin at the I-A junction so that titin's contribution to passive elasticity, and perhaps active contraction, can be measured. This versatile mouse model allows for a diverse set of biophysical experiments to elucidate the role of the giant protein titin in muscle function.

Single molecule force spectroscopy techniques have become important tools to study properties of proteins that operate under force; a common occurrence in Biology. Lack of specific attachment of single molecules to the force probe limits the measuring time and often leads to tethering at random places along a protein substrate. Here we present a technique based on polyprotein engineering and HaloTag attachment that is capable of avoiding these limitations. This method shows full-length polyprotein unfolding, high pick-up yield and detachment forces of ∼2 nN. Compared to other covalent attachments, this method shows a specific signature, given by the partial unfolding of HaloTag. We find that placing the HaloTag at the N-end of the construct shows an unfolding contour length of 66 nm and a mechanical strength of ∼131 pN. Placing the HaloTag at the C-end of the construct exposes to force a more stable part of the protein, which shows an increased mechanical strength of ∼491 pN and a contour length increment of 27 nm. We use HaloTag covalent attachment to study the folding of I27, a model protein from human titin. We expose I27 polyprotein constructs to successive cycles of high and low force, which unfolds and refolds the component protein domains. Covalent attachment greatly expands the tethering time of a polyprotein construct and allows for the measuring of the unfolding and folding rates from a single trace. Furthermore, repeated unfolding and refolding of the same polyprotein reveals subtle effects such as folding intermediates and slow oxidation of cysteines, which affects the mechanical stability of titin. This method opens a new approach to study protein folding at the human timescale, where proteins such as titin are slowly turned over after several days and to determine their energy landscape.

[EN]ADP-dependent glucokinases represent a unique family of kinases that belong to the ribokinase superfamily, being present mainly in hyperthermophilic archaea. For these enzymes there is no agreement about the magnitude of the structural transitions associated with ligand binding and whether they are meaningful to the function of the enzyme. We used the ADP-dependent glucokinase from Termococcus litoralis as a model to investigate the conformational changes observed in X-ray crystallographic structures upon substrate binding and to compare them with those determined in solution in order to understand their interplay with the glucokinase function. Initial velocity studies indicate that catalysis follows a sequential ordered mechanism that correlates with the structural transitions experienced by the enzyme in solution and in the crystal state. The combined data allowed us to resolve the open-closed conformational transition that accounts for the complete reaction cycle and to identify the corresponding clusters of aminoacids residues responsible for it. These results provide molecular bases for a general mechanism conserved across the ADP-dependent kinase family, This work was supported by Fondo Nacional de Desarrollo Científico y Tecnológico FONDECYT grant 1110137 to V.G, Spanish Ministry of Science and Innovation grants PET2008_0101, BIO2009-11184 and BFU2010-22260-C02-02 and the EC project ComplexINC (Framework Programme 7 (FP7) under grant agreement no. 279039) to M.C.V. The SAXS measurements (SAXS1 and SAXS2 beamlines) were supported by LNLS (Laboratorio Nacional de Luz Sincrotron), Sao Paulo, Brazil, to V.G. and J.A.R-P. We also thank the Departamento de Postgrado y Postitulo, Universidad de Chile by the support given to J.A.R-P to accomplish the SAXS studies in Sao Paulo. J.A.R-P and V.C-F are PhD fellowship from Comisión Nacional de Investigación Científica y Tecnológica de Chile. A.H-M. acknowledges the support of the PhD program in Molecular Biotechnology of the Universitat de Barcelona.

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. We engineered polyproteins based on the I27 domain of titin including two cysteine residues. Bifunctional crosslinking molecules specific for thiol groups were then used to generate intradomain covalent bridges between the engineered cysteines, much similar to the manner disulfides link polypeptide chains. Different bismaleimide reagents containing cleavable covalent bonds were tested for their ability to crosslink the I27 domain. We used single-molecule force- spectroscopy to pull from the modified polyproteins. Successfully crosslinked domains gave rise to lower increments in contour length after mechanical unfolding, consistent with the number of amino acids protected in the protein loop formed by the covalent bridge. Using our new strategy, virtually any covalent bond can be used to generate intradomain crosslinks in proteins. Our method can be adapted to produce hybrids between proteins and sugars, peptides or nucleic acids, which may be useful to study catalysis by enzymes such as amylases, cellulases, proteases or nucleases. This new generation of substrates holds the promise of becoming a key tool to study single-bond chemistry under force.

The elasticity of muscle tissue relies on tandem modular proteins such as titin, which gives muscles passive elasticity. A free-energy model is the only way to understand and predict the behavior under force of such complex proteins, composed of hundreds of individual domains. Here we use magnetic tweezers to measure the dynamics of single tandem modular proteins under constant force conditions during hour-long recordings. At forces between 4 to 17 pN we measure unfolding/refolding of individual domains as upward/downward steps in the end-to-end protein length, while higher forces yield unfolding steps exclusively. We find a strong force dependency of the step size of proteins undergoing folding/unfolding reactions with the applied force. This finding contradicts current free energy models of proteins that typically do not consider the polymeric nature of a denatured polypeptide chain under force and simply scale the free energy of a protein with the mechanical work. To explain the measured step size dependency with force we propose a new free energy model that also considers the entropic work needed to extend the molecule. Brownian dynamics simulations over the proposed free energy landscape accurately reproduce our experimental benchmarks. The experimental and theoretical advances demonstrated in this work provide a novel view on the free energy of proteins under force, now permitting a more realistic modeling of tissue elasticity.

I91 and I10 are archetypical Ig domains found in the giant muscle protein titin. Recent advances in magnetic tweezers force spectroscopy combined with HaloTag anchoring chemistry allow us to study I10 and I91 proteins at physiological forces (

Tensegrity is the property of tissues that allows them to regain their shape after a mechanical deformation. Constituent proteins support tensegrity by being able to generate a restoring force at any length. A salient feature of tissue aging is the oxidative modification of its proteins, thus compromising the tensegrity of the system (e.g. sagging skin). Here, we use magnetic tweezers to monitor the folding dynamics of single protein L molecules under force over times scales from hours to days. Mechanically unfolded proteins that are maintained extended for 22 hours entirely lose their ability to fold. This loss of folding is triggered by the exposure of the cryptic side chains to the oxidative environment, as it can be greatly slowed by adding an antioxidant to the solution. This phenomenon compromises the tensegrity of the protein by reducing its extensibility by 40%. We provide an analytical expression that describes the extensibility of a protein under force, combining the entropic elasticity of the polypeptide and the folding collapse. By incorporating an aging factor measured from the loss of protein folding over time, we can predict the loss of tensegrity. Our ability to accurately keep a single protein unfolded for hours to days presents a novel assay for accelerating aging. We anticipate that this will become a useful tool to discern the role of environmental contaminants to understand the loss of tensegrity in exposed tissues.

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.