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E.A. Kolobova, C.S. Klug, S.M. Buckheister, A.K. Kuznetzow, W.L. Hubbell, V.V. Gurevich; Identification of Elements Involved in Arrestin-rhodopsin Interaction by Site-directed Spin-labelling . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3176.
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Purpose: Site-directed spin-labeling/EPR was used to identify elements of arrestin and rhodopsin involved in their mutual interaction. Methods: Functionally active Cys-less mutants of arrestin and mutants of rhodopsin bearing a spin labeled side chain (R1) at selected positions were prepared. The mobility of each R1, determined from the EPR spectrum, was compared in the free proteins and in the arrestin-rhodopsin complex. Results: We have examined interaction of a truncated (residues 1-378) phosphorylation-independent arrestin mutant with both dark (Rh) and light activated (Rh*) rhodopsin containing R1 at either position 250 (on the inner surface of transmembrane helix 6, TM6, or 227 (on the outer surface of TM5). Light activation of Rh in the absence of arrestin leads to an increase and decrease in the mobility of 250 and 227, respectively. These changes are signatures for an outward displacement of TM6. Arrestin does not affect the EPR spectrum of dark Rh labeled at either position, or that of R1 at 227 in Rh*. In contrast, arrestin dramatically decreases the mobility of R1 at 250 in Rh*, suggesting that the internal binding cavity of rhodopsin is in one of arrestin docking sites. The spectrum of R1 at position 173 in "phosphorylation recognition" site of arrestin is complex with two components, one with limited mobility, in agreement with the crystal structure. In the presence of dark phosphorylated rhodopsin (P-Rh) the mobility of R1 at 173 is sharply reduced, indicative of the interaction of this site with receptor-attached phosphates even on inactive rhodopsin. In the presence of light-activated phosphorhodopsin (P-Rh*) the EPR spectrum reflects changes that can be interpreted in terms of a further reduction in mobility. Although the effect is less than that due to interaction with P-Rh, this result suggests involvement of this site in high-affinity binding. R1 at position 16 in arrestin (next to its second phosphate-binding element) is very dynamic and remains mobile in the presence of dark P-Rh and P-Rh*. Conclusions: Arrestin contacts the internal binding cavity of rhodopsin, which is also involved in transducin binding. The phosphate-binding element in arrestin beta-strand X (which includes position 173) directly interacts with P-Rh* and is highly immobilized in the complex. The introduction of spin labels into additional sites in both proteins will allow us to map out their interaction surfaces.
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