Titin is a giant protein that determines the elasticity of striated muscle and is thought to play important roles in numerous regulatory processes. Previous studies have shown that titin's PEVK domain interacts with F-actin, thereby creating viscous forces of unknown magnitude that may modulate muscle contraction. Here we measured, with optical tweezers, the forces necessary to dissociate F-actin from individual molecules of recombinant PEVK fragments rich either in polyE or PPAK motifs. Rupture forces at a stretch rate of 250 nm/s displayed a wide, nonnormal distribution with a peak at approximately 8 pN in the case of both fragments. Dynamic force spectroscopy experiments revealed low spontaneous off-rates that were increased even by low forces. The loading-rate dependence of rupture force was biphasic for polyE in contrast with the monophasic response observed for PPAK. Analysis of the molecular lengths at which rupture occurred indicated that there are numerous actin-binding regions along the PEVK fragments' contour, suggesting that the PEVK domain is a promiscuous actin-binding partner. The complexity of PEVK-actin interaction points to an adaptable viscoelastic mechanism that safeguards sarcomeric structural integrity in the relaxed state and modulates thixotropic behavior during contraction.

Here, we measured the concentrations of several ions in cultivated Gram-negative and Gram-positive bacteria, and analyzed their effects on polymer formation by the actin homologue MreB. We measured potassium, sodium, chloride, calcium and magnesium ion concentrations in Leptospira interrogans, Bacillus subtilis and Escherichia coli. Intracellular ionic strength contributed from these ions varied within the 130–273 mM range. The intracellular sodium ion concentration range was between 122 and 296 mM and the potassium ion concentration range was 5 and 38 mM. However, the levels were significantly influenced by extracellular ion levels. L. interrogans, Rickettsia rickettsii and E. coli MreBs were heterologously expressed and purified from E. coli using a novel filtration method to prepare MreB polymers. The structures and stability of Alexa-488 labeled MreB polymers, under varying ionic strength conditions, were investigated by confocal microscopy and MreB polymerization rates were assessed by measuring light scattering. MreB polymerization was fastest in the presence of monovalent cations in the 200–300 mM range. MreB filaments showed high stability in this concentration range and formed large assemblies of tape-like bundles that transformed to extensive sheets at higher ionic strengths. Changing the calcium concentration from 0.2 to 0 mM and then to 2 mM initialized rapid remodelling of MreB polymers.

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Gelsolin is a severing and capping protein that targets filamentous actin and regulates filament lengths near plasma membranes, contributing to cell movement and plasma membrane morphology. Gelsolin binds to the plasma membrane via phosphatidylinositol 4,5-bisphosphate (PIP2) in a state that cannot cap F-actin, and gelsolin-capped actin filaments are uncapped by PIP2 leading to filament elongation. The process by which gelsolin is removed from PIP2 at the plasma membrane is currently unknown. Gelsolin also binds ATP with unknown function. Here we characterize the role of ATP on PIP2-gelsolin complex dynamics. Fluorophore-labeled PIP2 and ATP were used to study their interactions with gelsolin using steady-state fluorescence anisotropy, and Alexa488-labeled gelsolin was utilized to reconstitute the regulation of gelsolin binding to PIP2-containing phospholipid vesicles by ATP. Under physiological salt conditions ATP competes with PIP2 for binding to gelsolin, while calcium causes the release of ATP from gelsolin. These data suggest a cycle for gelsolin activity. Firstly, calcium activates ATP-bound gelsolin allowing it to sever and cap F-actin. Secondly, PIP2-binding removes the gelsolin cap from F-actin at low calcium levels, leading to filament elongation. Finally, ATP competes with PIP2 to release the calcium-free ATP-bound gelsolin, allowing it to undergo a further round of severing.

Leiomodin proteins are vertebrate homologues of tropomodulin, having a role in the assembly and maintenance of muscle thin filaments. Leiomodin2 contains an N-terminal tropomodulin homolog fragment including tropomyosin-, and actin-binding sites, and a C-terminal Wiskott-Aldrich syndrome homology 2 actin-binding domain. The cardiac leiomodin2 isoform associates to the pointed end of actin filaments, where it supports the lengthening of thin filaments and competes with tropomodulin. It was recently found that cardiac leiomodin2 can localise also along the length of sarcomeric actin filaments. While the activities of leiomodin2 related to pointed end binding are relatively well described, the potential side binding activity and its functional consequences are less well understood. To better understand the biological functions of leiomodin2, in the present work we analysed the structural features and the activities of Rattus norvegicus cardiac leiomodin2 in actin dynamics by spectroscopic and high-speed sedimentation approaches. By monitoring the fluorescence parameters of leiomodin2 tryptophan residues we found that it possesses flexible, intrinsically disordered regions. Leiomodin2 accelerates the polymerisation of actin in an ionic strength dependent manner, which relies on its N-terminal regions. Importantly, we demonstrate that leiomodin2 binds to the sides of actin filaments and induces structural alterations in actin filaments. Upon its interaction with the filaments leiomodin2 decreases the actin-activated Mg2+-ATPase activity of skeletal muscle myosin. These observations suggest that through its binding to side of actin filaments and its effect on myosin activity leiomodin2 has more functions in muscle cells than it was indicated in previous studies.

Several kind of drugs—used in cancer treatments—such as cyclophosphamide (CP) can also trigger a disease classified as toxic polyneuropathy. Polyneuropathy is a simultaneous malfunction of several peripheral nerves, typical side effect of a cancer therapy. In our previous study, we used CP treated in vitro animal model (Guinea pig) with a comparable dosage and time handling of human protocol to show evidences of this drug-induced effects. We could show a dose-dependent difference between in Tm and ΔHcal of untreated and treated samples assigned to their intact muscle and nerve, blood plasma and red blood cells. In our current study we analyze this side effect on skeletal muscle actin (prepared from m. psoas of rabbit) by DSC (differential scanning calorimetry), to follow the possible consequence of drug treatment on the “activator” of muscle contraction. We have demonstrated that run of DSC curves, Tms together with the ΔHcal exhibit clear CP effect. In case of Ca2+ G actin it is manifested in a well separated second high denaturing temperature as a consequence of CP binding into the cleft. This way the nucleotide binding cleft with subdomains 1 and 3 becomes less flexible, indicating clear sensitivity to CP treatment. In F-actin samples, the main peak represents the thermal denaturation of subdomains 1 and 3, and the increased calorimetric enthalpy administrating Ca2+ as well as CP refers to a more rigid structure. These alterations can be the molecular background in the malfunction of muscle in case of polyneuropathy after CP treatment.

Titin is a giant filamentous protein traversing the half sarcomere of striated muscle with putative functions as diverse as providing structural template, generating elastic response, and sensing and relaying mechanical information. The Z-disk region of titin, which corresponds to the N-terminal end of the molecule, has been thought to be a hot spot for mechanosensing while also serving as anchorage for its sarcomeric attachment. Understanding the mechanics of titin's Z-disk region, particularly under the effect of binding proteins, is of great interest. Here we briefly review recent findings on the structure, molecular associations, and mechanics of titin's Z-disk region. In addition, we report experimental results on the dynamic strength of titin's Z1Z2 domains measured by nanomechanical manipulation of the chemical dimer of a recombinant protein fragment.

The actin is one of the main component of the eukaryotic cytoskeleton. The continuous rearrangement of actin filaments is provided by the different complexes with divalent cations (Ca2+ or Mg2+) and nucleotides (ATP, ADP). In the medical routine, cyclophosphamide (CP) is applied as cytostatic and it was shown that in vivo muscle filament system was changed by the CP treatment and it has direct interaction with actin monomers as well. The evolutionary importance of physical links between domains is one of the most interesting question to understand the multi-domain development of protein functions. Here, we analyse the thermal stability modifier act of inter-domain links in proteins, monitored by DSC, with the concept of that how did the nucleotide binding cleft between the two main domains of actin monomers affect the activation energy of domains if it was blocked or released by CP binding or dissociation, respectively. We investigated the importance of inter-domain linkers on the thermodynamic properties of actin. Ca2+ and Mg2+ bound G-actin can be stabilized by CP binding or polymerization. CP treatment of Ca2+-F actin lacks the structural integrity of the more flexible polymer and shows same stability as CP bound monomers. However, Mg2+-F actin did not show any kinetic response to the CP treatment. We can assume that the inter-domain linker of actin reduces the stability of the domains which leads to a more reactive and variable structure as a thermodynamic advantage for the development of a multi-domain protein can be blocked by CP treatment.