title: Rhodopsin photoproducts in 2D crystals.
abstract: The published electron microscope and X-ray structures of rhodopsin have made available a detailed picture of the inactive dark state of rhodopsin. Yet, the photointermediates of rhodopsin that ultimately lead to the activated receptor species still await a similar analysis. Such an analysis first requires the generation and characterization of the photoproducts that can be obtained in crystals of rhodopsin. We therefore studied with Fourier-transform infrared (FTIR) difference spectroscopy the photoproducts in 2D crystals of bovine rhodopsin in a p22(1)2(1) crystal form. The spectra obtained by cryotrapping revealed that in this crystal form the still inactive early intermediates batho, lumi, and meta I are similar to those obtained from rhodopsin in native disk membranes, although the transition from lumi to meta I is shifted to a higher temperature. However, at room temperature, the formation of the active state, meta II, is blocked in the crystalline environment. Instead, an intermediate state is formed that bears some features of meta II but lacks the specific conformational changes required for activity. Despite being unable to activate its cognate G protein, transducin, to a significant extent, this intermediate state is capable of interacting with functional transducin-derived peptides to a limited extent. Therefore, while unable to support formation of rhodopsin's active state meta II, 2D p22(1)2(1) crystals proved to be very suitable for determining 3D structures of its still inactive precursors, batho, lumi, and meta I. In future studies, FTIR spectroscopy may serve as a sensitive assay to screen crystals grown under altered conditions for potential formation of the active state, meta II.
authors.affiliation: Biophysics Group, Institut für Molekulare Medizin und Zellforschung, Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Str. 9, D-79104 Freiburg, Germany. reiner.vogel@biophysik.uni-freiburg.de
authors.forename: Reiner
authors.initials: R
authors.lastname: Vogel
authors.forename: Jonathan
authors.initials: J
authors.lastname: Ruprecht
authors.forename: Claudio
authors.initials: C
authors.lastname: Villa
authors.forename: Thorsten
authors.initials: T
authors.lastname: Mielke
authors.forename: Gebhard F X
authors.initials: GF
authors.lastname: Schertler
authors.forename: Friedrich
authors.initials: F
authors.lastname: Siebert
mesh_terms: D000818:Animals; D002417:Cattle; D003461:Crystallography; D019204:GTP-Binding Proteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D012243:Rhodopsin / Q000737:chemistry* / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature
chemical_list: D012243:Rhodopsin; D019204:GTP-Binding Proteins