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. Author manuscript; available in PMC: 2011 Jan 8.
Published in final edited form as: J Mol Biol. 2009 Oct 31;395(1):42–54. doi: 10.1016/j.jmb.2009.10.058

Fig. 3. Elimination of hydrophobic residues on the a-helix I/N-domain interface selectively reduces the stability of arrestin complex with P-Rh*.

Fig. 3

Binding of the indicated mutants to P-Rh* (A, B) or Rh* (C) was measured after the complex was allowed to dissociate for the indicated time at 4°C. Means +/− SD of three experiments performed in duplicate are shown. Half-life of the complex (t1/2) was determined using a single-exponential decay equation in GraphPad Prizm. T1/2 for curves shown in panel A for wild type (WT), h3A, h3A-2A, and 2A arrestin was 130±19, 171±17, 120±11**, and 77±14*** min, respectively (**, p<0.01, as compared to h3A; ***, p<0.001, as compared to WT). T1/2 for curves shown in panel B for 3A, h3A, 2A–3A, and 2A-3A-h3A arrestins was 9.1±1.4, 4.9±1.1**, 8.7±1.4, and 4.5±1.1** h, respectively (**, p<0.01, as compared to 3A and 2A–3A). T1/2 for curves shown in panel C for 3A, h3A, 2A–3A, and 2A-3A-h3A arrestins was 73±6, 77±9, 68±7, and 72±7 min, respectively (there were no statistically significant differences). The dissociation of mutants with reduced selectivity (B, C) was measured in the presence of 1 mM NH2OH, which chemically reacts with all-trans-retinal in light-activated rhodopsin, thereby preventing its re-binding to opsin. Abbreviations: 3A, F375A/V376A/F377A triple mutation; h3A, L103A/L107A/L111A triple mutation; 2A, V44A/L46A double mutation.