Zsolt Mártonfalvi

Different pressure–temperature behavior of the structured and unstructured regions of titin

Contrary to the classical view, according to which all proteins adopt a specific folded conformation necessary for their function, intrinsically unstructured proteins (IUPs) display random-coil-like conformation under physiological conditions. We compared the structured and unstructured domains from titin, a giant protein responsible for striated-muscle elasticity. A 171-residue-long fragment (polyE) of the disordered PEVK domain, and an Ig domain (I27) with ordered structure were investigated. FTIR (Fourier transform infrared) and fluorescence spectroscopy combined with a diamond anvil cell were used for investigation of the secondary structures under wide range of pressure and temperature. PolyE preserves its disordered characteristics across the entire range of investigated pressure (0-16kbar), temperature (0-100°C), pD (3-10.5) and different solvent conditions. The detailed temperature-pressure phase diagram of titin I27 was determined. At 30°C, increasing pressure unfolds titin I27 in one step at 10.5kbar. Increasing temperature at atmospheric pressure results in two transitions. At 50°C the secondary structure is loosened and the protein transforms into a molten-globule state. At 65°C the protein completely unfolds. Unfolding is followed by aggregation at ambient pressure. Moderate pressures (>2kbar), however, can prevent the protein from aggregation. Our experiments in wide range of physical parameters revealed four different structures for I27, while the unstructured character of the PEVK fragment is insensitive to these parameters.

Low-force transitions in single titin molecules reflect a memory of contractile history

ABSTRACT Titin is a giant elastomeric muscle protein that has been suggested to function as a sensor of sarcomeric stress and strain, but the mechanisms by which it does so are unresolved. To gain insight into its mechanosensory function we manipulated single titin molecules with high-resolution optical tweezers. Discrete, step-wise transitions, with rates faster than canonical Ig domain unfolding occurred during stretch at forces as low as 5 pN. Multiple mechanisms and molecular regions (PEVK, proximal tandem-Ig, N2A) are likely to be involved. The pattern of transitions is sensitive to the history of contractile events. Monte-Carlo simulations of our experimental results predicted that structural transitions begin before the complete extension of the PEVK domain. High-resolution atomic force microscopy (AFM) supported this prediction. Addition of glutamate-rich PEVK domain fragments competitively inhibited the viscoelastic response in both single titin molecules and muscle fibers, indicating that PEVK domain interactions contribute significantly to sarcomere mechanics. Thus, under non-equilibrium conditions across the physiological force range, titin extends by a complex pattern of history-dependent discrete conformational transitions, which, by dynamically exposing ligand-binding sites, could set the stage for the biochemical sensing of the mechanical status of the sarcomere.

Titin Domains Progressively Unfolded by Force Are Homogenously Distributed along the Molecule

Titin is a giant filamentous protein of the muscle sarcomere in which stretch induces the unfolding of its globular domains. However, the mechanisms of how domains are progressively selected for unfolding and which domains eventually unfold have for long been elusive. Based on force-clamp optical tweezers experiments we report here that, in a paradoxical violation of mechanically driven activation kinetics, neither the global domain unfolding rate, nor the folded-state lifetime distributions of full-length titin are sensitive to force. This paradox is reconciled by a gradient of mechanical stability so that domains are gradually selected for unfolding as the magnitude of the force field increases. Atomic force microscopic screening of extended titin molecules revealed that the unfolded domains are distributed homogenously along the entire length of titin, and this homogeneity is maintained with increasing overstretch. Although the unfolding of domains with progressively increasing mechanical stability makes titin a variable viscosity damper, the spatially randomized variation of domain stability ensures that the induced structural changes are not localized but are distributed along the molecules length. Titin may thereby provide complex safety mechanims for protecting the sarcomere against structural disintegration under excessive mechanical conditions.

Exclusion-Zone Dynamics Explored with Microfluidics and Optical Tweezers

The exclusion zone (EZ) is a boundary region devoid of macromolecules and microscopic particles formed spontaneously in the vicinity of hydrophilic surfaces. The exact mechanisms behind this remarkable phenomenon are still not fully understood and are debated. We measured the short- and long-time-scale kinetics of EZ formation around a Nafion gel embedded in specially designed microfluidic devices. The time-dependent kinetics of EZ formation follow a power law with an exponent of 0.6 that is strikingly close to the value of 0.5 expected for a diffusion-driven process. By using optical tweezers we show that exclusion forces, which are estimated to fall in the sub-pN regime, persist within the fully-developed EZ, suggesting that EZ formation is not a quasi-static but rather an irreversible process. Accordingly, the EZ-forming capacity of the Nafion gel could be exhausted with time, on a scale of hours in the presence of 1 mM Na2HPO4. EZ formation may thus be a non-equilibrium thermodynamic cross-effect coupled to a diffusion-driven transport process. Such phenomena might be particularly important in the living cell by providing mechanical cues within the complex cytoplasmic environment.

Individual Globular Domains and Domain Unfolding Visualized in Overstretched Titin Molecules with Atomic Force Microscopy

Titin is a giant elastomeric protein responsible for the generation of passive muscle force. Mechanical force unfolds titin’s globular domains, but the exact structure of the overstretched titin molecule is not known. Here we analyzed, by using high-resolution atomic force microscopy, the structure of titin molecules overstretched with receding meniscus. The axial contour of the molecules was interrupted by topographical gaps with a mean width of 27.7 nm that corresponds well to the length of an unfolded globular (immunoglobulin and fibronectin) domain. The wide gap-width distribution suggests, however, that additional mechanisms such as partial domain unfolding and the unfolding of neighboring domain multimers may also be present. In the folded regions we resolved globules with an average spacing of 5.9 nm, which is consistent with a titin chain composed globular domains with extended interdomain linker regions. Topographical analysis allowed us to allocate the most distal unfolded titin region to the kinase domain, suggesting that this domain systematically unfolds when the molecule is exposed to overstretching forces. The observations support the prediction that upon the action of stretching forces the N-terminal ß-sheet of the titin kinase unfolds, thus exposing the enzyme’s ATP-binding site and hence contributing to the molecule’s mechanosensory function.

Force generation by titin folding

Titin is a giant protein that provides elasticity to muscle. As the sarcomere is stretched, titin extends hierarchically according to the mechanics of its segments. Whether titins globular domains unfold during this process and how such unfolded domains might contribute to muscle contractility are strongly debated. To explore the force‐dependent folding mechanisms, here we manipulated skeletal‐muscle titin molecules with high‐resolution optical tweezers. In force‐clamp mode, after quenching the force (<10 pN), extension fluctuated without resolvable discrete events. In position‐clamp experiments, the time‐dependent force trace contained rapid fluctuations and a gradual increase of average force, indicating that titin can develop force via dynamic transitions between its structural states en route to the native conformation. In 4 M urea, which destabilizes H‐bonds hence the consolidated native domain structure, the net force increase disappeared but the fluctuations persisted. Thus, whereas net force generation is caused by the ensemble folding of the elastically‐coupled domains, force fluctuations arise due to a dynamic equilibrium between unfolded and molten‐globule states. Monte–Carlo simulations incorporating a compact molten‐globule intermediate in the folding landscape recovered all features of our nanomechanics results. The ensemble molten‐globule dynamics delivers significant added contractility that may assist sarcomere mechanics, and it may reduce the dissipative energy loss associated with titin unfolding/refolding during muscle contraction/relaxation cycles.

Topology of interaction between titin and myosin thick filaments

Titin is a giant protein spanning between the Z- and M-lines of the sarcomere. In the A-band titin is associated with the myosin thick filament. It has been speculated that titin may serve as a blueprint for thick-filament formation due to the super-repeat structure of its A-band domains. Accordingly, titin might provide a template that determines the length and structural periodicity of the thick filament. Here we tested the titin ruler hypothesis by mixing titin and myosin at in situ stoichiometric ratios (300 myosins per 12 titins) in buffers of different ionic strength (KCl concentration range 100-300 mM). The topology of the filamentous complexes was investigated with atomic force microscopy. We found that the samples contained distinct, segregated populations of titin molecules and myosin thick filaments. We were unable to identify complexes in which myosin molecules were regularly associated to either mono- or oligomeric titin in either relaxed or stretched states of the titin filaments. Thus, the electrostatically driven self-association is stronger in both myosin and titin than their binding to each other, and it is unlikely that titin functions as a geometrical template for thick-filament formation. However, when allowed to equilibrate configurationally, long myosin thick filaments appeared with titin oligomers attached to their surface. The titin meshwork formed on the thick-filament surface may play a role in controlling thick-filament length by regulating the structural dynamics of myosin molecules and placing a mechanical limit on the filament length.

Individual Globular Domains and Domain Unfolding Visualized in Overstretched Titin Molecules

Titin is a giant elastomeric protein responsible for the generation of passive muscle force. Mechanical force unfolds titins globular domains, but the exact structure of the overstretched titin molecule is not known. Here we analyzed, by using high-resolution atomic force microscopy, the structure of titin molecules overstretched with receding meniscus. The axial contour of the molecules was interrupted by topographical gaps with a mean width of 28.5 nm that corresponds well to the length of an unfolded globular (Ig or FN) domain. The wide and apparently multimodal gap-width distribution suggest, however, that additional mechanisms such as partial domain unfolding and the unfolding of neighboring domain multimers may also be present. In the folded regions we resolved globules with an average spacing of 5.9 nm, which is consistent with a titin chain composed globular domains with extended interdomain linker regions. Topographical analysis allowed us to allocate the most distal unfolded titin region to the kinase domain, suggesting that this domain systematically unfolds when the molecule is exposed to overstretching forces. The observations support the prediction that upon the action of stretching forces the N-terminal s-sheet of the titin kinase unfolds, thus exposing the enzymes ATP-binding site and hence contributing to the molecules mechanosensory function.