Biophys J, December 2001, p. 3285-3293, Vol. 81, No. 6

Rhodopsin-Transducin Interface: Studies with Conformationally Constrained Peptides

Rieko Arimoto,^* Oleg G. Kisselev, Gergely M. Makara,^* and Garland R. Marshall^*

 ^*Department of Biochemistry and Molecular Biophysics, Washington University, St. Louis, Missouri 63110, and  Department of Ophthalmology, St. Louis University, St. Louis, Missouri 63104 USA

To probe the interaction between transducin (G_t) and photoactivated rhodopsin (R*), 14 analog peptides were designed and synthesized restricting the backbone of the R*-bound structure of the C-terminal 11 residues of G_t derived by transferred nuclear Overhauser effect (TrNOE) NMR. Most of the analogs were able to bind R*, supporting the TrNOE structure. Improved affinities of constrained peptides indicated that preorganization of the bound conformation is beneficial. Cys347 was found to be a recognition site; particularly, the free sulfhydryl of the side chain seems to be critical for R* binding. Leu349 was another invariable residue. Both Ile and tert-leucine (Tle) mutations for Leu349 significantly reduced the activity, indicating that the Leu side chain is in intimate contact with R*. The structure of R* was computer generated by moving helix 6 from its position in the crystal structure of ground-state rhodopsin (R) based on various experimental data. Seven feasible complexes were found when docking the TrNOE structure with R* and none with R. The analog peptides were modeled into the complexes, and their binding affinities were calculated. The predicted affinities were compared with the measured affinities to evaluate the modeled structures. Three models of the R*/G_t complex showed strong correlation to the experimental data.

Biophys J, December 2001, p. 3285-3293, Vol. 81, No. 6
© 2001 by the Biophysical Society   0006-3495/01/12/3285/09  $2.00

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