Crowding alters F-actin secondary structure and hydration

Actin, a key component of the cytoskeleton in eukaryotic cells, plays a crucial role in regulating cell morphology and transport. The morphology, mechanical, and biochemical properties of these filaments and bundles are determined by their monomer structure and by protein-protein contacts. Crowded environments are known to organize filaments into bundles. However, less is known how crowding and bundling affect the structure of F-actin. Here, we employed two-dimensional infrared (2D IR) spectroscopy and structure-based spectral calculations to investigate the morphology-dependent secondary-structure and local environments in filaments and weakly or strongly bundled networks. The results indicate that actin undergo secondary structural changes upon bundling, resulting in a decrease in beta-sheet and increase in the loop conformations. Moreover, strongly bundled networks experience a decrease in backbone solvent exposure, with relatively low perturbation of alpha-helix and the beta-sheet nearly ``locked" in buried positions. Similar changes are observed in the loops, which become less hydrated but exhibit a dynamic environment. In summary, our study provides insights into the structure and structure-dependent local environment of actin biopolymers under morphology control by PEG, emphasizing the significance of loop structure as a critical player in actin network morphology and stability.