Edward C. Eckels

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.

Work Done by Titin Protein Folding Assists Muscle Contraction

Current 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.

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.

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.

DsbA is a switchable mechanical chaperone

In bacteria, many toxins and virulence factors pass through a translocon pore as unfolded polypeptides en route to the periplasm where they first encounter DsbA, a ubiquitous bacterial oxidoreductase enzyme that introduces disulphide bonds into nascent proteins. Here, using magnetic tweezers based single molecule force spectroscopy, we demonstrate for the first time that DsbA can also accelerate folding of cysteine-free proteins by using the globular domain of the protein L super-antigen as a substrate. This chaperone activity is tuned by the oxidation state of DsbA: oxidized DsbA is a strong promoter of folding, but the effect is weakened by reduction of the catalytic CXXC motif. We further localize the chaperone binding site of DsbA using a seven residue peptide which effectively blocks the foldase activity. DsbA assisted folding of proteins in the periplasm generates enough mechanical work to decrease the ATP consumption needed for periplasmic translocation by up to 33%. In turn, pharmacologic inhibition of this chaperone activity may open up a new class of anti-virulence agents.

CHAPTER 1.3:Real-time Detection of Thiol Chemistry in Single Proteins

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 carnivores 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 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.