title: Vibrational spectroscopy of bacteriorhodopsin mutants. Evidence that Thr-46 and Thr-89 form part of a transient network of hydrogen bonds.
abstract: The role of Thr-46 and Thr-89 in the bacteriorhodopsin photocycle has been investigated by Fourier transform infrared difference spectroscopy and time-resolved visible absorption spectroscopy of site-directed mutants. Substitutions of Thr-46 and Thr-89 reveal alterations in the chromophore and protein structure during the photocycle, relative to wild-type bacteriorhodopsin. The mutants T89D and to a lesser extent T89A display red shifts in the visible lambda max of the light-adapted states compared with wild type. During the photocycle, T89A exhibits an increased decay rate of the K intermediate, while a K intermediate is not detected in the photocycle of T89D at room temperature. In the carboxyl stretch region of the Fourier transform infrared difference spectra of T89D, a new band appears as early as K formation which is attributed to the deprotonation of Asp-89. Along with this band, an intensity increase occurs in the band assigned to the protonation of Asp-212. In the mutant T46V, a perturbation in the environment of Asp-96 is detected in the L and M intermediates which corresponds to a drop in its pK alpha. These data indicate that Thr-89 is located close to the chromophore, exerts steric constraints on it during all-trans to 13-cis isomerization, and is likely to participate in a hydrogen-bonding network that extends to Asp-212. In addition, a transient interaction between Thr-46 and Asp-96 occurs early in the photocycle. In order to explain these results, a previously proposed model of proton transport is extended to include the existence of a transient network of hydrogen-bonded residues. This model can account for the protonation changes of key amino acid residues during the photocycle of bacteriorhodopsin.
authors.affiliation: Department of Physics, Boston University, Massachusetts 02215.
authors.forename: K J
authors.initials: KJ
authors.lastname: Rothschild
authors.forename: Y W
authors.initials: YW
authors.lastname: He
authors.forename: S
authors.initials: S
authors.lastname: Sonar
authors.forename: T
authors.initials: T
authors.lastname: Marti
authors.forename: H G
authors.initials: HG
authors.lastname: Khorana
mesh_terms: D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006860:Hydrogen Bonding; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013055:Spectrophotometry, Infrared; D013816:Thermodynamics; D013912:Threonine; D014732:Vibration
chemical_list: D013912:Threonine; D001436:Bacteriorhodopsins