This research project aims at revealing the correlation between water structural changes in bacteriorhodopsin (bR) and its function, proton pumping. Fourier-transform infrared (FTIR) spectroscopy of mutant or isotope-labeled bR provided the following results.
(1) We constructed the IR measuring system to obtain "noise-free" difference spectra between bR and its intermediates. In addition, time-resolved FTIR,polarized FTIR,and detection of protein-protein interaction by FTIR are now possibly applied to retinal proteins, providing much more information on their structural changes.
(2) Location of water molecules in bR were examined by combination of bR mutants and FTIR.We have assigned the location of four water molecules, which are all present along the proton channel.
(3) A water molecule whose O-H stretching vibration is located at 3643 cm^<-1> changes H-bonding upon formation of the L-intermediate. We found that the water structural change is essential in the proton transfer from the retinal Schiff base to Asp85.
(4) We indentified Glu204 to be the proton release group. In addition, FTIR spectroscopy revealed hydrogen-bonding network between Asp85 and Glu204, including a water molecule in this region.
(5) We found that single amino acid substitution converts proton pumping bR to the chloride pump like halorhodopsin (hR). It is likely that both bR and hR share the common mechanism of ion-pumping and only a single amino acid determines the pump property.