Fig. 2.

VSFG spectra of the water–air interface and aqueous solutions of AFP-III of different concentration (0–90 μM). The VSFG spectra of AFP-III strongly depend on concentration. At concentrations below 10 μM (navy blue), the spectra are similar to the VSFG spectrum of water–air, except for the band at 3,600 cm⁻¹ that can be assigned to water molecules that are weakly hydrogen-bonded to the protein. A similar response is observed for other proteins (see Fig. S2). For concentrations >10 μM, the broad response in the OH stretch region disappears and a relatively narrow peak at 3,200 cm⁻¹ rises. This peak is assigned to water molecules contained in ice-like layers bound to the IBS of the AFP-III protein.