(a) SEC chromatogram monitoring absorbance at 280 nm (mAU) and estimated molecular mass (from MALS). (b) Apo-ABP (orange, open circle) exhibits a high initial fluorescence signal that is lowered in the presence of amantadine (orange, solid circle). As expected, 2LC3H6_13 (blue, open triangle) and 2LC3H6_13 plus amantadine (blue, solid triangle) exhibit a very low initial fluorescence signal and overlap almost identically. (c) The CD spectrum of ABP at 25°C, 75°C, 95°C, and 25°C after heating and cooling. The CD spectrum of ABP at 25°C suggests an all ɑ-helical structure that remains fairly stable up to 75°C. (d) 2D amide 1H-15N HMQC spectra (left) and 2D methyl 1H-13C HMQC spectra (right) of 250 μM apo-ABP (red) or ABP in the presence of 2 mM amantadine (blue) recorded at 800 MHz, 37°C. Titration of amantadine leads to significant changes in the ABP NMR spectra (arrows). To the right of the 2D methyl methyl 1H-13C HMQC spectra an inset of dissociation constant estimate through conventional fraction bound analysis is shown for the affected ILE methyl group, with an estimated KD of <55 μM. (e) NMR line shape fitting of ABP throughout the NMR titration with amantadine performed in the program TITAN using a two-state binding model for the affected ILE methyl group. The NMR data (gray) are shown versus the TITAN fit (magenta).