This title appears in the Scientific Report : 2009 
NMR-Lösungsstruktur der Loopregion Tyr$^{67}$ - Leu$^{77}$ des visuellen Arrestins im Komplex mit photoaktiviertem Rhodopsin
Feuerstein, Sophie (Corresponding author)
Strukturbiochemie; ISB-3
Jülich Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag 2009
XVI, 143 S.
Heinrich-Heine-Universität Düsseldorf, Diss., 2009
978-3-89336-571
Book
Dissertation / PhD Thesis
Funktion und Dysfunktion des Nervensystems
Schriften des Forschungszentrums Jülich. Reihe Gesundheit / Health 14
Hochschulschrift > Dissertation (Univ.)
OpenAccess
Please use the identifier: http://hdl.handle.net/2128/15182 in citations.


Rhodopsin, the visual pigment of rod outer segments of vertebrate photoreceptor cells, is the prototype of a G-protein coupled receptor (GPCR). Absorption of a single photon activates rhodopsin, which converts to a series of photointermediates. Metarhodopsin II (metaII) interacts with the G-protein transducin, thereby initiating the signaling cascade. The termination of the light response is accomplished by the phosphorylation of rhodopsin and the subsequent tight binding of arrestin to phosphorylated metaII. A sequential multi-site binding model for arrestin implicates engagement of two arrestin sites, the activation and the phosphorylation recognition sensor. Until now, there is no high resolution structure of transducin or arrestin in complex with light activated rhodopsin. The aim of this work was the structure determination of a tranducin or arrestin derived peptide in the rhodopsin bound state. Membrane embedded rhodopsin is not suitable for observation by liquid state NMR. However, transferred nuclear Overhauser effect (TrNOE) and transferred residual dipolar couplings (TrRDC) recorded on the free form of a peptide report on the membrane protein bound peptide conformation provided that there is sufficiently fast exchange between free and bound peptide. A library of synthetic peptides was screened for candidates that bind to metaII with an affinity that is in the range most suitable for TrNOE/TrRDC experiments. The peptides mimic previously identified binding regions from the G-protein transducin and the regulatory protein arrestin which interact with metaII in visual signal transduction. The loop region between $\beta$-strands V and VI of arrestin is currently discussed as part of the activation recognition site. TrNOE and TrRDC data showed that peptides which correspond to this loop region transiently bind to activated, unphosphorylated rhodopsin. TrNOE-derived proton-proton distances characterising the receptor-bound peptide were used as restraints in molecular dynamics-based simulated annealing protocols implemented in Xplor- NIH for structure calculation of the arrestin derived peptide Arr(67-77). Additionally, TrRDC data of uniformly $^{15}$N-labeled Arr(67-77) showed that the peptide seems to be appropriate for orientation determination, since $^{1}$J$_{NH}$ splittings measured directly after light activation showed the expected time dependent exponential decay. It was possible to solve the structure of an arrestin peptide in the receptor bound state by TrNOE data for the first time. The peptide Arr(67-77) reflects the loop region between $\beta$- strands V and VI of the parent arrestin molecule. In the crystal structure of inactive arrestin this region is unstructured and perhaps flexible. Binding to light activated rhodopsin induces an $\alpha$-helical structure between residues E$^{70}$ und M$^{75}$. The presented data indicate that Arr(67-77) most probably is part of the activation recognition sensor in the multi-site mechanism of arrestin-rhodopsin-binding.