Structural Requirements for the Stabilization of metarhodopsin II by the Cterminus of the alpha subunit of transducin

The retinal receptor rhodopsin undergoes a conformational change upon light excitation to form metarhodopsin II (Meta II), which allows interaction an d activation of its cognate G protein, transducin (G(t)). A C-terminal Il-a mino acid peptide from transducin, G(t alpha)-(340-350), has been shown to both bind and stabilize the Meta II conformation, mimicking heterotrimeric G,. Using a combinatorial library we identified analogs of G(t alpha)-(340- 350) that bound light-activated rhodopsin with high affinity (Martin, E. L. , Rens-Domiano, S., Schatz, P. J,, and Hamm, H. E. (1996) J. Biol. Chem. 27 1, 361-366). We have made peptides with key substitutions either on the bac kground of the native G(t alpha)-(340-350) sequence or on the high affinity sequences and used the stabilization of Meta II as a tool to determine whi ch amino acids are critical in G protein-rhodopsin interaction. Removal of the positive charge at the N termini by acylation or delocalization of the charge by K to R substitution enhances the affinity of the G(t alpha)-(340- 350) peptides for Meta II, whereas a decrease was observed following C-term inal amidation. Cys-347, a residue conserved in pertussis toxin-sensitive G proteins, was shown to interact with a hydrophobic site in Meta II. These studies provide further insight into the mechanism of interaction between t he G(t alpha) C terminus and light-activated rhodopsin.