Polyethylene glycol (PEG) 6 kDa and 20 kDa were purchased from Millipore Sigma (Product number 8.18897.1000 and 8.07491.1000). Potassium chloride (KCl), imidazole, dithiothreitol (DTT), magnesium chloride (MgCl 2 ), and Adenosine triphosphate (ATP) were purchased from Sigma-Aldrich. Trolox was purchased from Acros Organics. Actin 10/17 filament samples were prepared with a buffer concentration of 20 mM imidazole (pH 7.4), 50 mM potassium chloride (KCl), 2 mM magnesium chloride (MgCl 2 ), 1 mM dithiothreitol (DTT), 0.1 mM ATP, and 1 mM Trolox. All buffer solutions were prepared with deuterated water (D 2 O) to eliminate the strong water (OH) band in the amide I stretch region. The sample of actin bundles has an addition of 0.1% w/v (or 1 mg/mL) PEG with molecular weight of 6 kDa or 20 kDa to form a low or high degree of bundles, respectively, in buffer solutions. Note the mass per milliliter is equal for bundled samples, but the molecular weight between PEG molecules is different. Confocal microscopy revealed that a strongly bundled network occurs when the bundling degree is greater than 0.2 [a.u.] . The degree of bundling was shown to be below 0.2 for 0.1% PEG 6kDa, and above 0.2 for 0.1% PEG 20kDa . In this study, the PEG 6 kDa and 20 kDa samples are referred to as weakly and strongly bundled networks, respectively. All samples were freshly prepared, measured within 48 h, and stored at 4 • C before the measurements. The concentration of actin in all samples was adjusted to 1.5 mg/mL (36 µM) to produce a sufficiently high signal for 2D IR experiments but remained sufficiently low to remain within the biologically relevant regime. The actin samples are also below the concentration regime where actin organizes into nematic liquid .