In this study, we characterize the actin backbone structure and solvent environment upon morphological changes driven by polymer depletion. Actin filaments, weakly bundled networks, and strongly bundled networks are studied using two-dimensional infrared (2D IR) spectroscopy (Figure ) combined with theoretical modeling of residue participation in the vibrational states associated with specific frequency regions. The amide I mode along the backbone of actin (Figure ) is used as an infrared (IR) probe for the actin structure and local environments . The 2D IR spectra are interpreted using a vibrational model that relates amide I features to secondary structures, thus providing a molecular description of filament interactions within the different morphologies. In addition, the sub-picosecond dynamics of the solvent molecules are captured by the evolution of spectral lineshape as a function of pump and probe time delays. Results show that spectral features and timescales are similar in filaments and weakly bundled networks, but are very different for strongly bundled networks. Three secondary structures are assigned in the amide I frequency region: α-helix, β -sheet, and loops/turns. A small yet substantial conformational change from β -sheet to loop structure occurs upon the formation of strongly bundled networks. Further analysis of the solvent dynamics reveals the different effects of morphological changes on the local environments of the three different backbones. In summary, this work investigates the structure and dynamics of the actin backbone in different morphologies driven by polymer depletion, offering clues for the correlations between the structure and physical/biochemical properties of supramolecular biopolymer assemblies.