We investigate the backbone structure and structure-dependent solvent exposure using ultrafast laser spectroscopy and interpret the lineshape changes from filaments to bundles using structure-based vibrational models. The secondary structure changes derived from the amide I lineshape (Figure ) and the dynamics measured by 2D IR spectra (Figure ) can be used to understand the role of the actin backbone in the physical and biochemical functions of the actin networks. In general, filaments and weakly bundled networks (bundling degree lower than 0.2 ) share similar backbone structures and solvent environments, as evidenced by the similar amide I lineshape and dynamics across the two morphologies. However, the lineshape and dynamics for strongly bundled networks are different, suggesting changes in the local environment within this morphology. First, lineshape modeling indicates the structural transformation from β -sheet to loop/turn structure, although by a relatively small fraction. This finding is further strengthened by excitonic calculations and doorway state analysis (Figure and), where more pronounced loop features are accompanied by a decrease in the fraction of β -sheet in strongly bundled networks. Second, the increased features of the loops should demonstrate a more disordered or heterogeneous network in comparison to the filamentous sample, in agreement with dynamic-light-scattering measurements . Third, the strongly bundled network exhibits lower solvent exposure due to the interactions between neighboring filaments, leading to more constrained solvent environments and slower hydrogen bond exchange rates.