STRUCTURE AND FUNCTION IN RHODOPSIN - RHODOPSIN MUTANTS WITH A NEUTRAL AMINO-ACID AT E134 HAVE A PARTIALLY ACTIVATED CONFORMATION IN THE DARK STATE

The Glu-134-Arg-135 residues in rhodopsin, located near the cytoplasmi c end of the C helix, are involved in G protein binding, or activation , or both, Furthermore, the charge-neutralizing mutation Glu-134 to Gl n-134 produces hyperactivity in the activated state and produces const itutive activity in opsin, The Glu/Asp-Arg charge pair is highly conse rved in equivalent positions in other G protein-coupled receptors. To investigate the structural consequences of charge-neutralizing mutatio ns at Glu-134 and Arg-135 in rhodopsin, single spin-labeled side chain s were introduced at sites in the cytoplasmic domains of helices C (14 0), E (227), F (250), or G (316) to serve as ''molecular sensors'' of the local helix bundle conformation, In each of the spin-labeled rhodo psins, a Gin substitution was introduced at either Glu-134 or Arg-135, and the electron paramagnetic resonance spectrum of the spin label wa s used to monitor the structural response of the helix bundle, The res ults indicate that a Gin substitution at Glu-134 induces a photoactiva ted conformation around helices C and G even in the dark state, an obs ervation of potential relevance to the hyperactivity and constitutive activity of the mutant. In contrast, little change is induced in helix F, which has been shown to undergo a dominant motion upon photoactiva tion. This result implies that the multiple helix motions accompanying photoactivation are not strongly coupled and can be induced to take p lace independently, Gin substitution at Arg-135 produces only minor st ructural changes in the dark-or light-activated conformation, suggesti ng that this residue is not a determinant of structure in the regions investigated, although it may be functionally important.