title: Light-induced changes in the structure and accessibility of the cytoplasmic loops of rhodopsin in the activated MII state.
abstract: Bovine rhodopsin was specifically labeled on the cytoplasmic surface at cysteine 140 (the first residue of the loop connecting helices III and IV) or at cysteine 316 (in the loop connecting helix VII and the palmitoylation sites) with the fluorescent labels fluorescein and Texas Red. These loops are involved in activation and signal transduction. The time-resolved fluorescence depolarization was measured in the dark state and in the M(II) state, with labeled samples consisting of rhodopsin-octylglucoside micelles or rod outer segment (ROS) membranes. In this way the diffusional dynamics of the flexible loops of rhodopsin were measured for the first time directly on the nanosecond time scale. Control experiments showed that the large number of weak excitation pulses required in these single photon counting experiments leads to <5% bleaching of the sample. Rhodopsin was trapped in the activated M(II) state for the duration of the fluorescence experiments ( approximately 20 min) after illumination at pH 6 and 5 degrees C. For both types of samples and at both labeled positions the dynamics of the label and loop motion as monitored by the time constants of the depolarization were not significantly different in the two states of the receptor. The end-anisotropy increased, however, from 0.09 in the dark to 0.16 in the M(II) state for ROS samples labeled at C140. The corresponding numbers for the C316 position are 0.06 and 0.12. Light-induced activation in M(II) is thus associated with a large increase in the loop steric hindrance due to a changed loop domain structure on the cytoplasmic surface. These results are supported by fluorescence quenching experiments with I(-), which indicate a significant decrease in the collisional quenching constant k(q) and in accessibility in the M(II) state at both positions. The rotational correlation time of the rhodopsin micelles increased from 48 ns in the dark state to 60 ns in M(II). This increase is caused by a change in volume and/or shape and is consistent with a structural change. These results demonstrate that time-resolved fluorescence depolarization is a powerful tool to study the changes in conformation and dynamics of the cytoplasmic loops that accompany the activation of rhodopsin and other G-protein coupled receptors.
authors.affiliation: Biophysics Group, Department of Physics, Freie Universität Berlin, Arnimallee 14, D-14195 Berlin, Germany.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Mielke
authors.forename: Ulrike
authors.initials: U
authors.lastname: Alexiev
authors.forename: M
authors.initials: M
authors.lastname: Gläsel
authors.forename: H
authors.initials: H
authors.lastname: Otto
authors.forename: M P
authors.initials: MP
authors.lastname: Heyn
mesh_terms: D000818:Animals; D002417:Cattle; D003545:Cysteine / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry* / Q000378:metabolism; D019793:Fluorescein / Q000378:metabolism; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000378:metabolism; D008027:Light; D008823:Micelles; D010777:Photochemistry; D017433:Protein Structure, Secondary; D012243:Rhodopsin / Q000737:chemistry* / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014966:Xanthenes / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D008823:Micelles; D014966:Xanthenes; C034657:Texas red; D012243:Rhodopsin; D003545:Cysteine; D019793:Fluorescein