Protein function is coupled to its structural changes, for which stimulus-induced difference Fourier-transform infrared (FTIR) spectroscopy, is a powerful method. By optimizing the attenuated total reflection (ATR)-FTIR analysis on sodium-pumping rhodopsin KR2 in aqueous solution, we first measured the accurate difference spectra upon sodium binding in the whole IR region (4000-1000 cm(-1)). The new spectral window allows the analysis of not only the fingerprint region (1800-1000 cm(-1)) but also the hydrogen-bonding donor region (4000-1800 cm(-1)), revealing an unusually strong hydrogen bond of Tyr located in the sodium binding site of KR2. Progress in ATR-FTIR difference spectroscopy provides an approach to investigating stimulus-induced structural changes of membrane proteins under physiological aqueous conditions.