Time-dependent 2D IR spectra can be analyzed to evaluate the dynamics of the local environments around the actin secondary structure. The analysis of changes in 2D lineshapes as a function of the waiting time or pump-probe delay (t 2 ) can provide a measure of the correlation time in frequency fluctuations. Here, we use the center line slope method to represent changes in the 2D lineshapes following previous work by Kwak et al. , where the center line slope (CLS) is the slope of the ridge across the maxima of the amide I band. This slope typically decays exponentially with t 2 as the correlation between the excitation and detection frequencies is lost as a result of frequency fluctuation and energy redistribution among the normal modes. In general, the time constants extracted from the evolution of 2D lineshapes report on the dynamics or fluctuations of local environments within different secondary structures. Three frequency ranges were selected to characterize the dynamics of the different backbone structures based on PSA analysis and doorway state analysis in the amide I spectrum (Figure and Table ). The selected frequency ranges roughly match the ranges of R1, R3, and R5 in the doorway state analysis, where each frequency range covers the contribution primarily from one type of backbone structure. The center lines of the 2D IR spectra in the three frequency ranges are shown in the Supplementary Figure ). CLS versus t 2 is fitted with a single exponential decay of the form: