Exploring the Role of Neuropeptide PACAP in Cytoskeletal Function Using Spectroscopic Methods.

The behavior and presence of actin-regulating proteins are characteristic of various clinical diseases. Changes in these proteins significantly impact the cytoskeletal and regenerative processes underlying pathological changes. Pituitary adenylate cyclase-activating polypeptide (PACAP), a cytoprotective neuropeptide abundant in the nervous system and endocrine organs, plays a key role in neuron differentiation and migration by influencing actin. This study aims to elucidate the role of PACAP as an actin-regulating polypeptide, its effect on actin filament formation, and the underlying regulatory mechanisms. We examined PACAP27, PACAP38, and PACAP6-38, measuring their binding to actin monomers via fluorescence spectroscopy and steady-state anisotropy. Functional polymerization tests were used to track changes in fluorescent intensity over time. Unlike PACAP27, PACAP38 and PACAP6-38 significantly reduced the fluorescence emission of Alexa488-labeled actin monomers and increased their anisotropy, showing nearly identical dissociation equilibrium constants. PACAP27 showed weak binding to globular actin (G-actin), while PACAP38 and PACAP6-38 exhibited robust interactions. PACAP27 did not affect actin polymerization, but PACAP38 and PACAP6-38 accelerated actin incorporation kinetics. Fluorescence quenching experiments confirmed structural changes upon PACAP binding; however, all studied PACAP fragments exhibited the same effect. Our findings indicate that PACAP38 and PACAP6-38 strongly bind to G-actin and significantly influence actin polymerization. Further studies are needed to fully understand the biological significance of these interactions.

Spectroscopic characterization of the effect of mouse twinfilin-1 on actin filaments at different pH values.

The effect of mammalian twinfilin-1 on the structure and dynamics of actin filaments were studied with steady state fluorescence spectroscopy, total internal reflection fluorescence microscopy and differential scanning calorimetry techniques. It was proved before that the eukaryotic budding yeast twinfilin-1 can efficiently bind and severe actin filaments in vitro at low pH values. In the present work steady-state anisotropy measurements revealed that twinfilin can bind efficiently to F-actin. Dilution-induced depolymerization assay proved that mammalian twinfilin-1 has an actin filament severing activity. This severing activity was more pronounced at low pH values. Total internal reflection fluorescence microscopy measurements could support the severing activity of mouse twinfilin-1. The average rate of depolymerization was more apparent at low pH values. The differential scanning calorimetry measurements demonstrated that mammalian twinfilin-1 could reduce the stiffness within the actin filaments before the detachment of the actin protomers. The structural and dynamic reorganization of actin can support the twinfilin-1 induced separation of actin protomers. The measured data indicated that mammalian twinfilin-1 was able to accelerate the monomers dissociation and/or sever the filaments effectively at low pH values. It was concluded that twinfilin-1 can affect the F-actin in biological processes or under stress situations when the pH is markedly under the physiological level.

The effect of mouse twinfilin-1 on the structure and dynamics of monomeric actin.

The effect of twinfilin-1 on the structure and dynamics of monomeric actin was investigated with fluorescence spectroscopy and differential scanning calorimetry experiments. Fluorescence anisotropy measurements proved that G-actin and twinfilin-1 could form a complex. Due to the formation of the complexes the dissociation of the nucleotide slowed down from the nucleotide-binding pocket of actin. Fluorescence quenching experiments showed that the accessibility of the actin bound ε-ATP decreased in the presence of twinfilin-1. Temperature dependent fluorescence resonance energy transfer and differential scanning calorimetry experiments revealed that the protein matrix of actin becomes more rigid and more heat resistant in the presence of twinfilin-1. The results suggest that the nucleotide binding cleft shifted into a more closed and stable conformational state of actin in the presence of twinfilin-1.

The effect of toxofilin on the structure and dynamics of monomeric actin.

The effects of toxofilin (an actin binding protein of Toxoplasma gondii) on G-actin was studied with spectroscopy techniques. Fluorescence anisotropy measurements proved that G-actin and toxofilin interact with 2:1 stoichiometry. The affinity of toxofilin to actin was also determined with a fluorescence anisotropy assay. Fluorescence quenching experiments showed that the accessibility of the actin bound ε-ATP decreased in the presence of toxofilin. The results can be explained by the shift of the nucleotide binding cleft into a closed conformational state. Differential scanning calorimetry measurements revealed that actin monomers become thermodynamically more stable due to the binding of toxofilin.

Dynamic strength of titin's Z-disk end.

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.