Figure 3.

ATR-FTIR spectroscopic measurements of amide-proton exchange in the rmMBP-F-actin assemblies. (A) Spectra for the dry film before soaking with D₂O (black curve) and at 6 h after adding D₂O (red curve). Amide I and II bands for both fully isotopically labeled rmMBP and unlabeled actin (two major bands for β-and α-structures are observed) in the protonated state, as well as amide I′ and II′ bands for the deuterated fractions of both rmMBP and actin, are indicated. (B) A series of FTIR spectra at different time points after adding D₂O as indicated. The spectra in both panels A and B are normalized to the integrated peak intensity of the amide I band. The amide II′ band for deuterated rmMBP increases very rapidly within the first second of the time course, as compared to the increase in the corresponding band in actin. Later on, the intensity of the rmMBP amide II′ band very quickly reaches near-saturation, whereas that of the actin amide II′ band continues to increase almost linearly. (C) The absorbance as a function of time for deuterated rmMBP amide II′ (red curve), deuterated actin amide II′ (green curve), and deuterated rmMBP amide I′ (blue curve). The solid lines are the global exponential fitting results, whereas the scattered points are the experimental values of the absolute absorbance. The points are not normalized. The initial drop in intensity is due to the swelling effect on the addition of D₂O to the dry film.