Light-Induced Two-Dimensional FT-IR Spectroscopy of BacterioRhodopsin

Two-dimensional Fourier transform infrared (2D FT-IR) spectroscopy was applied to study the slower states of the membrane protein bacterioRhodopsin (bR) photocycle, with bR adsorbed on a ZnSe attenuated total reflectance (ATR) crystal. The M and the N states of the bR photocycle could be discriminated, and 2D spectra revealed an N state absorption band at 1536 cm−1 that is normally covered by the broader and bigger 1527 cm−1 M state absorption band. 2D FT-IR absorbance changes are presented in a more convenient way for analysis than in traditional 2D FT-IR spectroscopy by using 2D-phase spectra and phase/amplitude calculations.