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S.A. Vishnivetskiy, R. Wiener, A. Lvov, J.A. Hirsch, V.V. Gurevich; Temperature Dependence of Arrestin Binding to Rhodopsin: Role of Arrestin Structural Elements in Stabilizing its Basal Conformation . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1516.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To elucidate the relative role of arrestin structural elements in the stabilization of its basal (inactive) state. Methods: We measured the rate of wild type (WT) and mutant arrestin binding to light-activated unphosphorylated (Rh*) and phosphorylated (P-Rh*) rhodopsin at various temperatures. The binding data were correlated with the estimates of arrestin flexibility obtained from temperature-dependent changes of CD spectra. Results: The level of arrestin binding measured in a standard 5 min. assay reflects the on-rate of the process. WT arrestin binding to its preferred target, P-Rh*, peaks at physiological temperature (37-40oC). Less than 5% of the peak binding is observed at 0oC, and less than 50% at 20oC, consistent with very high arrestin activation energy. Arrestin binding to Rh* follows the same pattern. Two main intramolecular interactions keep arrestin in its basal state: a network of charged residues in the polar core localized between the two arrestin domains and three-element interaction between the C-tail, beta-strand I, and alpha-helix I. We disrupted these two interactions by R175E (RE) and F375A,V376A,F377A (3A) mutations, respectively. Both mutants at 20oC bind essentially as well as at 37oC. At 0oC, RE, 3A, and RE+3A mutants demonstrate 60%, 90%, and 90% of peak binding, respectively, suggesting that both mutations significantly lower activation barrier, 3A being more effective than RE. Interestingly, temperature dependence of the binding of the three phosphorylation-independent mutants to Rh* qualitatively resembles the binding of WT arrestin to P-Rh*, suggesting that even constitutively active mutants still need a certain "push" to assume active conformation. CD spectra at different temperatures correlate well with binding data and show that 3A and RE mutants at lower temperatures are as flexible as WT arrestin at substantially higher temperatures. Conclusions: The necessity to disrupt main intramolecular interactions in arrestin explains its high activation energy, which can be lowered (but cannot be eliminated entirely) by pre-disrupting these interactions by targeted mutations.
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