Water structural changes at the proton uptake site (the Thr46-Asp96 domain) in the L intermediate of bacteriorhodopsin.
Fourier transform infrared spectra of the L intermediate of light-adapted bacteriorhodopsin were examined for recombinant proteins with amino acid substitutions at Thr46 and Asp96. Two O-H stretching vibrational bands of water, at 3607 and 3577 cm-1, change into stronger H-bonding states in L of the wild type. Thr46-->Val substitution abolished these bands in spite of the fact that [3-18O]threonine-labeling did not shift them, indicating that they correspond to coordination of the water with Thr46. The two water bands were restored, although with changed frequencies, by an additional Asp96-->Asn substitution in Thr46-->Val/Asp96-->Asn. A single Asp96-->Asn substitution abolished the 3607 cm-1 band. Thus, Asp96 also takes part in structural changes in water. The perturbations of these water molecules in the L intermediate displayed a weak correlation with the ratio of intensity change in the two vibrational bands of the Schiff base mode at 1312 and 1301 cm-1 and the rate for the deprotonation of the Schiff base at the L-to-M reaction of the photocycle. We find, therefore, that the water molecules in the cytoplasmic Asp96-Thr46 domain, which comprises the site of proton uptake after formation of the M intermediate, undergo structural changes in the L intermediate already. These changes are transmitted to the extracellular domain and affect interaction of the Schiff base with Asp85, that is far removed from this region.
Yamazaki
Y
Y
Department of Biophysics, Faculty of Science, Kyoto University, Japan.
Hatanaka
M
M
Kandori
H
H
Sasaki
J
J
Karstens
W F
WF
Raap
J
J
Lugtenburg
J
J
Bizounok
M
M
Herzfeld
J
J
Needleman
R
R
D001224:Aspartic Acid / Q000737:chemistry*; D001436:Bacteriorhodopsins / Q000737:chemistry*; D006860:Hydrogen Bonding; D008027:Light; D010103:Oxygen Isotopes; D011487:Protein Conformation; D011522:Protons; D012545:Schiff Bases / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D013912:Threonine / Q000737:chemistry*; D014867:Water / Q000737:chemistry*
D010103:Oxygen Isotopes; D011522:Protons; D012545:Schiff Bases; D014867:Water; D013912:Threonine; D001224:Aspartic Acid; D001436:Bacteriorhodopsins