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Qiuyan Chen, Tiandi Zhuang, Sergey A. Vishnivetskiy, Min-Kyu Cho, Tarjani M. Thaker, Tina M. Iverson, Vsevolod V. Gurevich, Charles R. Sanders; NMR Study of Arrestin-1 Binding to Rhodopsin. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4132.
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The arrestin-receptor interaction is a complex multi-step process. Arrestins undergo global conformational changes upon binding to their cognate receptors, but the conformation of active, receptor-bound arrestins remains unknown. We identified arrestin-1 elements engaged by different functional forms of rhodopsin and the conformational changes induced by receptor binding.
We used solution nuclear magnetic resonance (NMR) spectroscopy of 15N-labeled arrestin-1, free and in the presence of light activated (Rh*), phosphorylated light activated rhodopsin (P-Rh*), and phospho-opsin (P-Ops) in bicelles.
Solution NMR was used to assign ~40% of arrestin-1 backbone resonances. Native rhodopsin-containing disc membranes are too large for NMR, whereas detergents that solubilize rhodopsin denature arrestin-1. Bicelles, where bilayered lipid discs are edge-stabilized by detergent, provide a native membrane-like environment for rhodopsin and preserve arrestin-1 structure and function, as judged by near-UV circular dichroism spectra and arrestin binding to P-Rh*, Rh*, and P-Ops. We reconstituted different functional forms of rhodopsin into the bicelles and collected the spectra of arrestin-1 bound to Rh*, P-Rh*, and P-Ops. To identify changes in binding-induced chemical shifts, NMR spectra of free arrestin-1 were compared with the spectra collected in the presence of increasing amounts of Rh*, P-Rh*, and P-Ops. To make the arrestin-1-P-Rh* complex amenable to NMR analysis, its affinity was reduced using elevated NaCl. Arrestin elements engaged by Rh*, P-Rh*, and P-Ops were identified. The results suggest that distinct arrestin-1 residues are engaged by Rh* and P-Rh*. The data demonstrate that arrestin-1 binds P-Ops and P-Rh* similarly, which is consistent with the crystal structure of opsin resembling the activated form of rhodopsin.
Rh*, P-Rh*, and P-Ops engage multiple arrestin-1 residues and induce distinct conformational rearrangements in the arrestin-1 molecule. Bicelles provide a viable alternative to HDL nanoparticles for biophysical studies of arrestin interactions with G protein-coupled receptors.NIH grants EY011500, GM077561, GM081756 (VVG), GM080513 (CRS), EY018435, GM095633 (TMI).
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