Local and distant protein structural changes on photoisomerization of the retinal in bacteriorhodopsin

The photoisomerization of the retinal in bacteriorhodopsin is selective and efficient and yields perturbation of the protein structure within femtosec onds. The stored light energy in the primary intermediate is then used for the net translocation of a proton across the membrane in the microsecond to millisecond regime. This study is aimed at identifying how the protein cha nges on photoisomerization by using the O-H groups of threonines as interna l probes. Polarized Fourier-transform IR spectroscopy of [3-O-18]threonine- labeled and unlabeled bacteriorhodopsin indicates that 3 of the threonines (of a total of 18) change their hydrogen bonding. One is exchangeable in D2 O, but two are not. A comprehensive mutation study indicates that the resid ues involved are Thr-89, Thr-17, and Thr-121 (or Thr-90). The perturbation of only three threonine side chains suggests that the structural alteration at this stage of the photocycle is local and specific. Furthermore, the st ructural change of Thr-17, which is located >11 A from the retinal chromoph ore, implicates a specific perturbation channel in the protein that accompa nies the retinal motion.