No abstract

Arrestins are regulatory molecules for G-protein coupled receptor function. In visual rhodopsin, selective binding of arrestin to the cytoplasmic side of light-activated, phosphorylated rhodopsin (P-Rh*) terminates signaling via the G-protein transducin. While the "phosphate-sensor" of arrestin for the recognition of receptorattached phosphates is identified, the molecular mechanism of arrestin binding and the involvement of receptor conformations in this process are still largely hypothetic. Here we used fluorescence pump-probe and time-resolved fluorescence depolarization measurements to investigate the kinetics of arrestin conformational changes and the corresponding nanosecond dynamical changes at the receptor surface. We show that at least two sequential conformational changes of arrestin occur upon interaction with P-Rh*, thus providing a kinetic proof for the suggested multistep nature of arrestin binding. At the cytoplasmic surface of P-Rh*, the structural dynamics of the amphipathic helix 8 (H8), connecting transmembrane helix 7 and the phosphorylated C-terminal tail, depends on the arrestin interaction state. We find that a high mobility of H8 is required in the low-affinity (prebinding) but not in the high-affinity binding state. High-affinity arrestin binding is inhibited when a bulky, inflexible group is bound to H8, indicating close interaction. We further show that this close steric interaction of H8 with arrestin is mandatory for the transition from prebinding to high-affinity binding; i.e., for arrestin activation. This finding implies a regulatory role for H8 in activation of visual arrestin, which shows high selectivity to P-Rh* in contrast to the broad receptor specificity displayed by the two nonvisual arrestins. membrane receptor | protein conformational change | binding kinetics

We studied functional interaction structures of the vertebrate membrane photoreceptor rhodopsin containing retinal as a chromophore. Using time-resolved fluorescence depolarization we analyzed real-time dynamics and conformational changes of the cytoplasmic helix 8 (H8) preceding the long C-terminal tail of rhodopsin. H8 runs parallel to the membrane surface and extends from transmembrane helix 7 whose highly conserved NPxxY(x)F motif connects that region of rhodopsin with the retinal binding pocket. Our measurements indicate that photo-induced retinal isomerization from 11-cis to all-trans provokes conformational changes of H8, including slower motion and reduced flexibility, that are specific for the active metarhodopsin-II photo-intermediate. These conformational changes are absent in the retinal-devoid state opsin and in the phosphorylated metarhodopsin-II state upon receptor deactivation. Furthermore we show that membrane rim effects can influence interfacial reactions at the cytoplasmic rhodopsin surface such as proton transfer reactions between surface and aqueous bulk phase or binding of the signaling protein transducin visualized with single-molecule widefield microscopy. These findings are important for an understanding of the effects of membrane structure on the photo-transduction mechanism.

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.