Fluctuations in the local environment of the actin backbone are primarily driven by hydrogen bonding, either to other residues in the proteins or to water molecules. Because water molecules undergo fast dynamics on a sub-picosecond timescale , backbone fluctuations are faster in solvent-exposed regions than in residues that are locked in more rigid secondary structures, such as the protein core. Using waiting-time-dependent 2D IR spectra, we construct an evaluation of the dynamics timescale by analyzing the changes in lineshapes as a function of the waiting time or pump-probe delay (t 2 ). As described in the Methods section, we use the center line slope method to represent changes in the 2D lineshapes and extract the dynamics of local environments for three secondary structures in different frequency regions: α-helix, β -sheet, and loops (Supplementary Figure ) Figure . Analysis of the vibrational dynamics for each secondary structure indicates less hydration around the actin backbone in the strongly bundled network. The center line slope (CLS) is plotted versus the delay time (t 2 ) between the pump and probe pulses. The solid lines represent exponential fitting curves, as described in the text. Light-to-dark colors represent filaments in strongly bundled network samples.