It is evident that more than one band appears along the diagonal in the amide I stretch region, as shown in Figure . All samples exhibit a strong band at 1650 cm -1 and a weaker band at 1630 cm -1 . In addition, the 2D IR lineshape of strongly bundled networks is different from that of filaments or weakly bundled networks. The broad amide I lineshape is known to arise from multiple bands positioned at different frequencies because of the different backbone conformations, where α-helix appears around 1650 cm -1 , β -sheet around 1630 cm -1 , and loops around 1680 cm -171, 74, 75 . In Figure , the peak shape in weakly bundled networks does not change significantly compared to the filament, suggesting similar secondary structures between the filament and the network with a low degree of bundling. In contrast, in the spectra of the strongly bundled network, the low-frequency band appears less prominent, while a broader shoulder arises on the blue frequency side compared with the filament and weakly bundled network samples. The lineshape difference of the strongly bundled network sample corresponds to backbone conformational changes and solvent exposure changes under the depletion of PEG, where actin filaments form a high degree of bundles.