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

Heterotrimeric G-proteins interact with their G-protein-coupled receptors (GPCRs) via key binding elements comprising the receptor-specific C-terminal segment of the alpha-subunit and the lipid anchors at the alpha-subunit N-terminus and the gamma-subunit C-terminus. Direct information about diffusion and interaction of GPCRs and their G-proteins is mandatory for an understanding of the signal transduction mechanism. By using single-particle tracking, we show that the encounters of the alpha-subunit C-terminus with the GPCR rhodopsin change after receptor activation. Slow as well as less restricted diffusion compared to the inactive state within domains 60-280 nm in length was found for the receptor-bound C-terminus, indicating short-range order in rhodopsin packing.

The cytoplasmic surface of G protein-coupled receptors plays a central role for activation and deactivation of the receptor. To understand the molecular mechanisms which underlie these processes, we determined the surface charge density and its changes upon activation directly at the cytoplasmic surface of bovine rhodopsin and correlated these changes with key events in receptor activation. The surface charge density was calculated from the ionic strength dependence of the apparent pK(a) of the surface-bound pH-indicator dye fluorescein according to the Gouy-Chapman theory. The surface charge density at pH 6.5 changes by 0.8 +/- 0.2 elementary charge/1000 A(2) in rod outer segment disk membranes and by 0.4 +/- 0.2 elementary charge/1000 A(2) in rhodopsin/dodecylmaltoside micelles upon formation of the active metarhodopsin-II state. By comparison of these surface charge density values determined with and without the native lipid environment, we calculated the charge change to about 1 elementary charge/cytoplasmic rhodopsin surface. The more positive surface charge density in metarhodopsin-II decreases back to the dark state level of sigma = -2.0 +/- 0.2 elementary charges/1000 A(2) in the opsin state, providing further evidence that the cytoplasmic surface properties after metarhodopsin-II decay resemble almost those of the dark state.

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A nowhere-zero k-flow is an assignment of edge directions and integer weights in the range 1, . . , , k -1 to the edges of an undirected graph such that at every vertex the flow in is equal to the flow out. Tutte has conjectured that every bridgeless graph has a nowhere-zero 5-flow. We show that a counterexample to this conjecture, minimal in the class of graphs embedded in a surface of fixed genus, has no face-boundary of length <7. Moreover, in order to prove or disprove Tutte's conjecture for graphs of fixed genus y , one has to check graphs of order at most 28(y -1) in the orientable case and 14(y -2) in the nonorientable case. So, in particular, it follows immediately that every bridgeless graph of orientable genus 5 1 or nonorientable genus 5 2 has a nowhere-zero 5-flow. Using a computer, we checked that all graphs of orientable genus 5 2 or nonorientable genus 5 4 have a nowhere-zero 5-flow.