Study of the binding environment of α-factor in its G protein-coupled receptor using fluorescence spectroscopy.

Mating in Saccharomyces cerevisiae is induced by the interaction of α-factor (W¹H²W³L[sup 4]Q[sup 5]L[sup 6]K[sup 7]P[sup 8]G[sup 9]Q[sup 10]P[sup 11]M[sup 12]Y[sup 13]) with its cognate G protein-coupled receptor (Ste2p). Fifteen fluorescently labeled analogs of α-factor in which the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group was placed at the αN-terminus and in side-chains at positions 1, 3, 4, 6, 7, 12 and 13 were synthesized and assayed for biological activity and receptor affinity. Eleven of the analogs retained 6-60% of the biological activity of the α-factor, as judged using a growth arrest assay. The binding affinities depended on the position of NBD attachment in the peptide and the distance of the tag from the backbone. Derivatization of the positions 3 and 7 side-chains with the NBD group resulted in analogs with affinities of 17-35% compared with that of α-factor. None of the other NBD-containing agonists had sufficient receptor affinity or strong enough emission for fluorescence analysis. The position 3 and 7 analogs were investigated using fluorescence spectroscopy and collisional quenching by KI in the presence of Ste2p in yeast membranes. The results showed that the λ[sub max] of NBD in the position 7 side-chain shifted markedly to the blue (510 nm) when separated by 4 or 6 bonds from the peptide backbone and that this probe was shielded from quenching by KI. In contrast, separation by 3, 5, 10 or more bonds resulted in λ[sub max] (≈540 nm) and collisional quenching constants consistent with increasing degrees of exposure. The NBD group in the position 3 side-chain was also found to be blue shifted (λ[sub max]-520 nm) and shielded from solvent. These results indicate that the position 7 side-chain is likely interacting with a pocket formed by extracellular domains of Ste2p, whereas the side-chain of Trp³ is in a hydrophobic pocket possibly within the transmembrane... [ABSTRACT FROM AUTHOR]