title: Structural changes in bacteriorhodopsin caused by two-photon-induced photobleaching.
abstract: Bacteriorhodopsin (BR) is the key protein of the halobacterial photosynthetic system. BR assembles into two-dimensional crystalline patches, the so-called purple membranes (PM), and acts as a light-driven proton pump converting light energy into the chemical energy of a proton gradient over the cell membrane. The two-photon absorption (TPA) of BR is so far not fully understood. Astonishingly high TPA cross sections have been reported, but the molecular mechanisms have not been elucidated. In this work, we address structural changes in BR and PM upon TPA, investigating its TPA photochemistry by spectroscopy, small-angle X-ray scattering, as well as electron and atomic force microscopy. We observe that TPA of BR leads to formation of an UV-absorbing N-retinyl-bacterioopsin state, which is accompanied by the loss of crystalline order in PM. FTIR and CD spectroscopy confirm that BR trimers as well as the secondary structure of the BR molecules are preserved. We demonstrate that excitation by TPA results in the photochemical reduction of the retinal Schiff base, which in turn causes a permanent asymmetric shape change of BR, similar to the one transiently observed during the photocycle-related opening and closing of the cytoplasmic proton half channel. This shape change causes PM sheets to merely roll up toward the extracellular side and causes the loss of crystallinity of PM. We present a model for the TPA photoresponse of BR, which also explains the irreversibility of the process in terms of a photochemical reduction of the Schiff base.
authors.affiliation: Department of Structural Biology, Max-Planck-Institute of Biophysics, Max-von-Laue-Str. 3, D-60438 Frankfurt, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Rhinow
authors.forename: Martin
authors.initials: M
authors.lastname: Imhof
authors.forename: Ivan
authors.initials: I
authors.lastname: Chizhik
authors.forename: Roelf-Peter
authors.initials: RP
authors.lastname: Baumann
authors.forename: Norbert
authors.initials: N
authors.lastname: Hampp
mesh_terms: D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000648:ultrastructure; D002942:Circular Dichroism; D003460:Crystallization; D019617:Halobacterium salinarum / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D038761:Photobleaching; D017785:Photons; D017433:Protein Structure, Secondary; D018717:Purple Membrane / Q000737:chemistry* / Q000648:ultrastructure; D053838:Scattering, Small Angle; D017550:Spectroscopy, Fourier Transform Infrared; D014961:X-Ray Diffraction
chemical_list: D001436:Bacteriorhodopsins