Transducin (T), the heterotrimeric guanine nucleotide binding protein in rod outer segments, serves as an intermediary between the receptor protein, rhodopsin, and the effector protein, cGMP phosphodiesterase. Labeling of T with dansyl chloride (DnsCl) inhibited its light-dependent guanine nucleotide binding activity. Conversely, DnsCl had no effect on the functionality of rhodopsin. Approximately 2-3 mol of DnsCl were incorporated per mole of T. Since fluoroaluminate was capable of activating DnsCl-modified T, this lysine-specific labeling compound did not affect the guanine nucleotide-binding pocket of T. However, the labeling of T with DnsCl hindered its binding to photoexcited rhodopsin, as shown by sedimentation experiments. Additionally, rhodopsin completely protected against the DnsCl inactivation of T. These results demonstrated the existence of functional lysines on T that are located in the proximity of the interaction site with the photoreceptor protein.

Transducin (T), a GTP-binding protein involved in phototransduction of rod photoreceptor cells, is a heterotrimer arranged as two units, the alpha-subunit (T alpha) and the beta gamma-complex (T beta gamma). The role of the carboxyl groups in T was evaluated by labeling with N,N'-dicyclohexylcarbodiimide (DCCD) and 1-ethyl 3-(3-dimethylaminopropyl) carbodiimide (EDC). Only a minor effect on the binding of beta, gamma-imido guanosine 5'-triphosphate (GMPpNp) to T was observed in the presence of the hydrophobic carbodiimide, DCCD. Similarly, the GMPpNp binding activity of the reconstituted holoenzyme was not significantly affected when T alpha was combined with DCCD-treated T beta gamma. However, the binding of guanine nucleotides to the reconstituted T was approximately 50% inhibited when DCCD-labeled T alpha was incubated with T beta gamma. In contrast, treatment of T with the hydrophilic carbodiimide, EDC, completely impaired its GMPpNp-binding ability. EDC-modified T was incapable of interacting with illuminated rhodopsin, as determined by sedimentation experiments. However, rhodopsin only partially protected against the inactivation of T. Additionally, analyses of trypsin digestion patterns showed that fluoroaluminate was not capable of activating the EDC-labeled T sample. The function of the reconstituted holoenzyme was also disrupted when EDC-modified T alpha was combined with T beta gamma, and when EDC-treated T beta gamma was incubated with T alpha.

Mutation of L125R in trasmembrane helix III of rhodopsin, associated with the retinal degenerative disease retinitis pigmentosa, was previously shown to cause structural misfolding of the mutant protein. Also, conservative mutations at this position were found to cause partial misfolding of the mutant receptors. We report here on a series of mutations at position 125 to further investigate the role of Leu125 in the correct folding and function of rhodopsin. In particular, the effect of the size of the substituted amino‐acid side chain in the functionality of the receptor, measured as the ability of the mutant rhodopsins to activate the G protein transducin, has been analysed. The following mutations have been studied: L125G, L125N, L125I, L125H, L125P, L125T, L125D, L125E, L125Y and L125W. Most of the mutant proteins, expressed in COS‐1 cells, showed reduced 11‐cis‐retinal binding, red‐shifts in the wavelength of the visible absorbance maximum, and increased reactivity towards hydroxylamine in the dark. Thermal stability in the dark was reduced, particularly for L125P, L125Y and L125W mutants. The ability of the mutant rhodopsins to activate the G protein transducin was significantly reduced in a size dependent manner, especially in the case of the bulkier L125Y and L125W substitutions, suggesting a steric effect of the substituted amino acid. On the basis of the present and previous results, Leu125 in transmembrane helix III of rhodopsin, in the vicinity of the β‐ionone ring of 11‐cis‐retinal, is proposed to be an important residue in maintaining the correct structure of the chromophore binding pocket. Thus, bulky substitutions at this position may affect the structure and signallling of the receptor by altering the optimal conformation of the retinal binding pocket, rather than by direct interaction with the chromophore, as seen from the recent crystallographic structure of rhodopsin.