Identifying Functionally Important Conformational Changes in Proteins: Activation of the Yeast α-factor Receptor Ste2p

摘要

We have developed a procedure in which disulfide crosslinks are used to identify regions of proteins that undergo functionally important intramolecular motion. The approach was applied to the identification of disulfide bonds that stabilize the active state of the yeast α-mating pheromone receptor Ste2p, a member of the superfamily of G Protein Coupled Receptors. Cysteine residues were introduced at random positions in targeted regions of a starting allele of Ste2p that completely lacks cysteines. Libraries of mutated receptors were then screened for alleles that exhibit constitutive signaling. Two strongly activated alleles were recovered containing cysteine residues in transmembrane segments 5 and 6. Constitutive activity of these alleles was dependent on the presence of both introduced cysteines and was sensitive to reducing agent. Crosslinked peptides derived from the mutant receptors were detected by immunoblotting. Additional sites of crosslinking between transmembrane segments 5 and 6 that did not lead to constitutive activation were also identified. These results indicate that relative motion of the transmembrane segments 5 and 6 in the extracellular half of the membrane is sufficient to activate the receptor and that transmembrane segment 6, but not transmembrane segment 5, exhibits rotational mobility that is not associated with receptor activation.