Chemical Modification of Transducin with Iodoacetic Acid: Transducin-α Carboxymethylated at Cys347 Allows Transducin Binding to Light-Activated Rhodopsin but Prevents Its Release in the Presence of GTP

Modification of transducin (T) with iodoacetic acid (IAA) inhibited its light-dependent guanine nucleotide-binding activity. Approximately 1 mol of [3H]IAA was incorporated per mole of T. Cys347, located on the α-subunit of T (Tα), was identified as the major labeled residue in the [3H]IAA-modified holoenzyme. In contrast, Cys135 and Cys347 were modified with [3H]IAA in the isolated Tα. IAA-modified T was able to bind tightly to photoexcited rhodopsin (R*), but GTP did not promote the dissociation of the complex between alkylated T and R*. In addition, R* protected against the inhibition of T by IAA. A comparable inactivation of T and analogous interactions between T and R* were observed when 2-nitro 5-thiocyanobenzoic acid (NTCBA) was used as the modifying reagent (J. O. Ortiz and J. Bubis, 2001, Effects of differential sulfhydryl group-specific labeling on the rhodopsin and guanine nucleotide binding activities of transducin, Arch. Biochem. Biophys. 387, 233–242). However, while carboxymethylated T was capable of liberating GDP in the presence of R*, NTCBA-modified T was unable to release the guanine nucleotide diphosphate upon incubation with the photoactivated receptor. Thus, IAA-labeling stabilized a T:R* complex intermediate carrying the empty nucleotide pocket conformation of T. On the other hand, NTCBA-modified T seemed to be “locked” in the GDP-bound state of T, even in the presence of R*.