Fig. 1.

Sum frequency generation (SFG) spectra. The ssp (denoting s-, s-, and p-polarized sum-frequency output, visible input, and infrared input, respectively) SFG spectra of the peptides of amide A and amide I modes, sketch for the hydration of amide I mode, and the relationship between hydrogen–deuterium exchange (HDX) ratio and amide I frequency. a The ssp SFG spectra of the peptides in the N-H region. The spectra in black curves are measured at lipid bilayer/H₂O interface while the ones in red curves are measured at lipid bilayer/D₂O interface after 5-h HDX experiments. b The ssp amide I SFG spectra of peptides at lipid/H₂O interface. The solid curves in  a, b are the fitting curves using Supplementary Eq. 1. c Sketch for the carbonyl groups forming a bifurcated H bonding to water molecules upon hydration. d Fitting amplitude ratio $\left({\chi }_{\mathrm{5h}\mathrm{HDX}}^{\left(2\right)}∕{\chi }_{\mathrm{before}\mathrm{HDX}}^{\left(2\right)}\right)$ of the ssp SFG spectra in the N-H region after 5-h HDX and before HDX against the amide I frequency. The vertical error bars represent experimental error in determining the amplitude ratio from the fitting procedure (see Supplementary Note 2). The horizontal error bars indicate the standard error of the mean of the fitting center frequency from five independent spectra