title: Water structural changes at the proton uptake site (the Thr46-Asp96 domain) in the L intermediate of bacteriorhodopsin.
abstract: 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.
authors.affiliation: Department of Biophysics, Faculty of Science, Kyoto University, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Yamazaki
authors.forename: M
authors.initials: M
authors.lastname: Hatanaka
authors.forename: H
authors.initials: H
authors.lastname: Kandori
authors.forename: J
authors.initials: J
authors.lastname: Sasaki
authors.forename: W F
authors.initials: WF
authors.lastname: Karstens
authors.forename: J
authors.initials: J
authors.lastname: Raap
authors.forename: J
authors.initials: J
authors.lastname: Lugtenburg
authors.forename: M
authors.initials: M
authors.lastname: Bizounok
authors.forename: J
authors.initials: J
authors.lastname: Herzfeld
authors.forename: R
authors.initials: R
authors.lastname: Needleman
mesh_terms: 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*
chemical_list: D010103:Oxygen Isotopes; D011522:Protons; D012545:Schiff Bases; D014867:Water; D013912:Threonine; D001224:Aspartic Acid; D001436:Bacteriorhodopsins