Application of FTIR Spectroscopy to the Structural Study on the Function of Bacteriorhodopsin

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Abstract
Difference FTIR spectroscopy of the photointermediates of bacteriorhodopsin is informative for changes in H‐bonding, the protonation states, and the bond orientation of functional residues such as C=O, N‐H, and O‐H of the chromophore, protein residues, peptide bonds, and internal water molecules. The vibrational bands of the chromophore are found at frequencies similar to those observed by resonance Raman spectra. Moreover, FTIR gives clear results on the N— in‐plane bending vibration and C14–C15 stretching vibrational modes, both of which are useful for the analysis of the structure of the chromophore. The protonation states and H‐bonding changes of intramembrane protonated aspartic acid residues can be revealed only by FTIR spectroscopy, in combination with proper isotope labeling and site‐directed mutagenesis. The results with Asp‐96 and Asp‐85 in the L, M, and N photointermediates were especially useful for understanding the proton transfer mechanism. Besides amino acid groups in the protein, peptide C=O vibrations were assigned to a specific bond by the use of site‐directed isotope labeling. Water molecules which undergo structural changes upon photoreaction were attributed specifically to those interacting with particular protein residues by mutational studies. On the basis of these FTIR studies, the role of the L intermediate is emphasized as the key intermediate that creates the conditions for the proton transfer reaction from the Schiff base to Asp‐85 and subsequent proton uptake reactions in the cytoplasmic domain.

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