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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.

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.

Background There is an emerging field to put into practice new strategies for developing molecules with antimicrobial properties. In this line, several metals and metalloids are currently being used for these purposes, although their cellular effect(s) or target(s) in a particular organism are still unknown. Here we aimed to investigate and analyze Au3+ toxicity through a combination of biochemical and molecular approaches. Results We found that Au3+ triggers a major oxidative unbalance in Escherichia coli, characterized by decreased intracellular thiol levels, increased superoxide concentration, as well as by an augmented production of the antioxidant enzymes superoxide dismutase and catalase. Because ROS production is, in some cases, associated with metal reduction and the concomitant generation of gold-containing nanostructures (AuNS), this possibility was evaluated in vivo and in vitro. Conclusions Au3+ is toxic for E. coli because it triggers an unbalance of the bacterium’s oxidative status. This was demonstrated by using oxidative stress dyes and antioxidant chemicals as well as gene reporters, RSH concentrations and AuNS generation.

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.

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.

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.

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.

Thiol chemistry provides a way for proteins to alter their form and function rapidly and reversibly. Although a variety of bulk techniques have been developed to ascertain the oxidation state and bonding of cysteine thiols, these methods may destroy the sample or lead to unwanted side reactions. Single-molecule force spectroscopy harnesses the ability to track protein folding and unfolding pathways with angstrom precision to detect changes in thiol chemistry in a real-time and non-destructive manner. As the oxidation state of the thiol changes, owing to intramolecular disulfide bonding or post-translational modification, changes to the protein topology and stability can be detected by unfolding of single-protein domains using the atomic force microscope. Not only does this provide a means to probe the mechanism of covalent bond scission by small nucleophiles and enzymes, but also a tool by which to monitor the activity of single oxidoreductase molecules as they introduce and rearrange disulfide bonds while protein substrates fold. Although a carnivore's bite damages tissue by tearing apart molecular bonds, nature has provided enzymatic machinery to repair the bonds, a process that can be directly observed using single-molecule techniques.

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

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.

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.