title: Dissection of environmental changes at the cytoplasmic surface of light-activated bacteriorhodopsin and visual rhodopsin: sequence of spectrally silent steps.
abstract: The physico-chemical properties as well as the conformation of the cytoplasmic surface of the 7-helix retinal proteins bacteriorhodopsin (bR) and visual rhodopsin change upon light activation. A recent study found evidence for a transient softening of bR in its key intermediate M [Pieper et al. (2008) Phys. Rev. Lett. 100, 228103] as a direct proof for the functional significance of protein flexibility. In this report we compare environmental and flexibility changes at the cytoplasmic surface of light-activated bR and rhodopsin detected by time-resolved fluorescence spectroscopy. The changes in fluorescence of covalently bound fluorescent probes and protein real-time dynamics were investigated. We found that in fluorescently labeled bR and rhodopsin the intensity of fluorescein and Atto647 increased upon formation of the key intermediates M and metarhodopsin-II, respectively, suggesting different surface properties compared to the dark state. Furthermore, time-resolved fluorescence anisotropy experiments reveal an increase in steric restriction of loop flexibility because of changes in the surrounding protein environment in both the M-intermediate as well as the active metarhodopsin-II state. The kinetics of the fluorescence changes at the rhodopsin surface uncover multiple transitions, suggesting metarhodopsin-II substates with different surface properties. Proton uptake from the aqueous bulk phase correlates with the first transition, while late proton release seems to parallel the second transition. The last transition between states of different surface properties correlates with metarhodopsin-II decay.
authors.affiliation: Department of Physics, Freie Universität Berlin, Berlin, Germany.
authors.forename: Tai-Yang
authors.initials: TY
authors.lastname: Kim
authors.forename: Martina
authors.initials: M
authors.lastname: Moeller
authors.forename: Kathrin
authors.initials: K
authors.lastname: Winkler
authors.forename: Kristina
authors.initials: K
authors.lastname: Kirchberg
authors.forename: Ulrike
authors.initials: U
authors.lastname: Alexiev
mesh_terms: D001436:Bacteriorhodopsins / Q000737:chemistry*; D003593:Cytoplasm / Q000737:chemistry* / Q000528:radiation effects*; D019617:Halobacterium salinarum / Q000737:chemistry; D007700:Kinetics; D008027:Light; D012243:Rhodopsin / Q000737:chemistry*; D013053:Spectrophotometry; D013997:Time Factors
chemical_list: D001436:Bacteriorhodopsins; D012243:Rhodopsin