Investigating the biophysical impact of Electromagnetic Fields on protein structures: a comparative analysis between Tubulin and G-actin

The cytoskeleton, composed primarily of microtubules and actin filaments, plays a crucial role in maintaining cellular architecture, regulating intracellular transports and supporting dynamic processes. Beyond their structural roles, cytoskeletal proteins also contribute to electrostatic regulation and can act as pathways for intracellular bioelectric signalling. Understanding how external physical stimuli modulate these proteins is therefore of biological and therapeutic interest. This thesis investigated the effects of electromagnetic field (EMF) stimulation on tubulin and G-actin using complementary biophysical approaches. Dynamic Light Scattering (DLS) was used to monitor hydrodynamic diameter and zeta potential, turbidity assays assessed polymerization dynamics, Raman spectroscopy analysed molecular vibrations and conductivity measurements proved electrostatic and charge transport properties. Two light-based devices with distinct physical characteristics were tested: Bioptron, emitting polarized broad-spectrum light, and Vielight, delivering near-infrared stimulation. DLS revealed device- and protein-specific changes in hydrodynamic diameter under colloidally stable conditions. For tubulin dimers, the diameter was largely preserved under Vielight, while Bioptron induced modest decreases and a slight broadening of the distribution with a reduction in zeta potential, suggesting weak clustering. In G-actin, Bioptron increased the hydrodynamic diameter and broadened the size distribution, consistent with light-induced remodelling or oligomerization, while Vielight produced only minor and uniform shifts. These changes were accompanied by zeta potential variations: Bioptron reduced the negative charge of G-actin, favouring aggregation, while Vielight caused subtler modifications. Raman spectroscopy of both proteins did not show some characteristic peaks, such as the amide I band around 1650 cm⁻¹, and stimulated and control samples were nearly identical, indicating that Bioptron irradiation did not measurably affect their vibrational profiles. Conductivity assays, performed under buffer conditions optimized for charge transport, provided complementary information. Tubulin showed low intrinsic conductivity and only modest changes upon irradiation. Under diluted buffer conditions, a further decrease in conductivity was observed, consistent with the reduced ionic strength and charge transport. G-actin exhibited high baseline conductivity, attributable to its acidic surface, but showed marked losses after Bioptron stimulation and moderate changes with Vielight. Turbidity assays, performed exclusively on tubulin, demonstrated a temporary acceleration of polymerization immediately after irradiation, followed by reduced reassembly after storage, particularly under Bioptron. Overall, these findings reveal that tubulin and G-actin display distinct sensitivities to EMF stimulation and can be selectively modulated according to wavelength, polarization, and concentration. This modulation may be relevant in diseases involving cytoskeletal dysfunction, including Alzheimer’s disease, depression, and actin-related disorders. In conclusion, this study shows that external light stimulation can influence the structural stability and electrostatic properties of cytoskeletal proteins. While these results open perspectives for photobiomodulation as a therapeutic strategy, further in vitro and in vivo studies are needed to validate these findings and determine their clinical applicability.