Computational Multiscale Modelling to Shed Light into Actin Isoforms Distinctive Properties

Actin is one of the structural and functional units of the cytoskeleton and has a pivotal role in the maintenance of cell shape and motility, thanks to its capability of assembling into filaments, which are referred to as actin microfilaments or F-actin to distinguish this form from the unassembled globular G-actin. In particular, proper cell functioning depends on the dynamic and mechanical properties of F-actin. Therefore, abnormalities in the mechanisms of assembly may lead to the development of numerous pathologies. Actin belongs to a protein family that includes six different isoforms that share a remarkably high sequence similarity but differ in tissue expression and cellular localization. In this context, one of the most studied isotypes is the a isotype, which composes the F-actin microfilaments at a skeletal muscle level and is implicated in muscle contraction together with myosin. On the other side, the ß isotype mainly forms the actin microfilaments of the cytoskeleton, performing functions related to the motility of the cell and the maintenance of its shape. Although the distinct amino-acid sequences and functions performed by the a and ß variants of actin are known, there is still reduced evidence regarding the difference between them in terms of the relationship between the atomic-level properties of G-actin and the macroscopical properties of the resulting microfilament. Given the fundamental role of actin inside the cell and the identification of different pathologies related to abnormalities in F-actin, the study of the structural, dynamic, and mechanical differences in the microfilaments formed by the two isotypes provide useful information to understand how subtle differences in the amino acid sequence are related to differences in the macroscopic properties of the filament, paving the way for the treatment of pathologies related to genetic alterations in actin isoforms. In this context, computational molecular modeling tools, such as Molecular Dynamics (MD), are employed to inspect the atomic-level properties of protein assemblies and relate them to macroscopic evidence. Therefore, the objective of this work is to exploit computational molecular modeling to characterize a and ß actin isoforms in their filamentous forms, providing insights into both the atomic-level properties and macroscopic mechanical properties of the resulting microfilaments. In particular, representative portions of a and ß actin microfilaments were reconstructed starting from available experimental data and then analyzed through MD simulations. Moreover, the actin microfilaments with lengths of hundreds of nanometers were modeled and studied using Elastic Network Modeling-based Normal Mode Analysis (NMA) to derive their mechanical properties from the obtained vibration frequencies, following an approach previously proposed in the literature. The results suggest that the two actin isoforms are characterized by different residue-level flexibilities. Furthermore, the two actin variants were characterized by a different amount of contact surface area between monomers within filaments, a property that reflects in differences in terms of mechanical properties as predicted by NMA. Overall, our results suggested that computational tools can provide useful insights into actin molecular and macroscopic properties, representing an important starting point to understand how subtle changes in the amino acid sequence can alter the properties of supramolecular assemblies.