Rafael Tapia-Rojo

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.

Trigger factor chaperone acts as a mechanical foldase

Proteins fold under mechanical forces in a number of biological processes, ranging from muscle contraction to co-translational folding. As force hinders the folding transition, chaperones must play a role in this scenario, although their influence on protein folding under force has not been directly monitored yet. Here, we introduce single-molecule magnetic tweezers to study the folding dynamics of protein L in presence of the prototypical molecular chaperone trigger factor over the range of physiological forces (4–10 pN). Our results show that trigger factor increases prominently the probability of folding against force and accelerates the refolding kinetics. Moreover, we find that trigger factor catalyzes the folding reaction in a force-dependent manner; as the force increases, higher concentrations of trigger factor are needed to rescue folding. We propose that chaperones such as trigger factor can work as foldases under force, a mechanism which could be of relevance for several physiological processes.Proteins fold under mechanical force during co-translational folding at the ribosome. Here, the authors use single molecule magnetic tweezers to study the influence of chaperones on protein folding and show that the ribosomal chaperone trigger factor acts as a mechanical foldase by promoting protein folding under force.

Proteins Breaking Bad: A Free Energy Perspective

Protein aging 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 aging. 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 naive 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.

Disulfide bonds: the power switches of elastic proteins

The delivery of mechanical power, a crucial component of animal motion, is constrained by the universal compromise between force and velocity of its constituent molecular systems1,2. Here we demonstrate a switchable power amplifier in the elastic proteins that comprise contractile tissues. Elastic proteins are typically very large and composed of tandem repeats of individually folded domains3–5, which unfold and extend under force and readily refold when the force is quenched. Cryptic cysteine residues are common in elastic proteins like titin where they can oxidize to form intra-domain disulfide bonds, limiting the extensibility of an unfolding domain6–8. However, the functional significance of disulfide-bonds in elastic proteins remains unknown and may be fundamental to tissue mechanics. Here we use ultra-stable magnetic tweezers force spectroscopy9,10 to study the elasticity of a disulfide bonded modular titin protein operating in the physiological range, with the ability to control the oxidation state of the protein in real time. We show that the midpoint folding probability of the parent Ig domain reversibly shifts from 4.0 pN to 12.8 pN upon oxidation. In this force range, the folding contraction dominates the elastic recoil of the protein, delivering stepwise mechanical work which depends on the oxidation state in an all-or-none manner. For example, the output power of a folding contraction at 6 pN goes from 0 zW to 6,000 zW upon introduction of the disulfide bond. This large amount of power is delivered by folding at forces where single molecular motors are typically stalled. We explain our results with a simple polymer model where the extensibility of the protein is determined in a binary form by the presence or absence of the disulfide bond. Our results demonstrate, for the first time, the functional significance of disulfide bonds as potent power amplifiers in proteins operating under force.The delivery of mechanical power, a crucial component of animal motion, is constrained by the universal compromise between force and velocity of its constituent molecular systems. Here we demonstrate a switchable power amplifier in an Ig domain of the massive muscle protein titin. Titin is composed of many tandem repeats of individually foldable Ig domains, which unfold and extend during muscle stretch and readily refold when the force on titin is quenched during a contraction. Cryptic cysteine residues are common in elastic proteins like titin where they can oxidize to form intra-domain disulfide bonds, limiting the extensibility of an unfolding domain. However, the functional significance of disulfide-bonds in titin Ig domains remains unknown and may be fundamental to muscle mechanics. Here we use ultra-stable magnetic tweezers force spectroscopy to study the elasticity of a disulfide bonded modular titin protein operating in the physiological range, with the ability to control the oxidation state of the protein in real time using both organic reagents and oxidoreductase enzymes. We show that presence of an oxidized disulfide bond allows the parent Ig domain to fold at much higher forces, shifting the midpoint folding probability from 4.0 pN to 12.8 pN after formation. The presence of disulfide bonds in titin regulates the power output of protein folding in an all-or-none manner, providing for example at 6.0 pN, a boost from 0 to 6,000 zeptowatts upon oxidation. At this same force, single molecular motors such as myosin are typically stalled and perform little to no work. We further demonstrate that protein disulfide isomerase (PDI) readily reintroduces disulfide bonds into unfolded titin Ig domains, an important mechanism for titin which operates under a resting force of several pN in vivo. Our results demonstrate, for the first time, the functional significance of disulfide bonds as potent power amplifiers in titin and provide evidence that protein folding can generate substantial amounts of power to supplement the myosin motors during a contraction.

A Magnetic Head Design for Manipulating Single Proteins with Force

Magnetic heads are ubiquitously used to record and read on magnetic tapes in technologies as diverse as old cassettes or VHS tapes, modern hard drive disks, or magnetic bands in credit/debit or subway cards. They are designed to convert electric signals into fluctuations on the magnetic field at very high frequencies, crucial for the high density storage which is demanded nowadays. Here, we twist this traditional use of magnetic heads and implement one in a new force spectrometer design, where the magnetic field is used to pull on proteins tethered to superparamagnetic beads. Our instrument offers the same features as magnetic tweezers (intrinsic force-clamp conditions, with accurate control of the force, and intrinsic stability), but with the novel ability of changing the force instantaneously, which allows to investigate protein dynamics at very short timescales, or under arbitrary force signals. We calibrate our instrument by relying on Karlqvist approximation of the field created by a magnetic head (the first building block of magnetic recording theory) through the force scaling of the unfolding/folding step-sizes of protein L, used as a molecular template. This results in a force range between 0 and 50 pN, when working at distances above 250 μm, and electric currents up to 1 A. We illustrate the potential of our instrument by studying the folding mechanism of protein L upon ultra-fast force quenches. This allows us to describe that, in a short timescale of 50 ms, the unfolded protein L evolves to an ensemble of weak collapsed states, eventually acquiring the native conformation in a timescale of seconds. Our instrumental development provides a unique capability of interrogating individual molecules under fast-changing force signals, which opens a range of novel force spectroscopy schemes of unexplored biological significance.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 new 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 for the first time the capability of interrogating individual molecules under fast-changing forces with a control and resolution below a fraction of a pN, opening a range of novel force spectroscopy protocols to study protein dynamics under force.

Isopeptide-Blocker Impairs the Mechanics of Recently Translated Pilin Proteins

Gram-positive bacteria, such as Streptococcus pyogenes, 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 protein from unfolding. Here, we designed a peptide to intervene in the formation of the isopeptide bond on the pilin Spy0128 from Streptococcus pyogenes, preventing folding and rendering the Spy0128 susceptible to protease digestion. By using a combination of protein engineering and single-molecule force spectroscopy, we demonstrate that the isopeptide-blocker peptide interferes with the formation of the isopeptide bond. While the intact Spy0128 is inextensible under mechanical forces, the intervened Spy0128 is completely extensible and lacks of mechanical stability. We propose that this isopeptide-blocker affords a novel strategy for mechanically-targeted antibiotics which, by blocking the folding structure of bacterial pili, could prevent the colonization of infectious microorganisms.ABSTRACT 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. Significance At 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.

Molecular strategy for blocking isopeptide bond formation in nascent pilin proteins

Significance At 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. 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.

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.