The isolation of mutations affecting the stabilities of transmembrane proteins is useful for enhancing the suitability of proteins for structural characterization and identification of determinants of membrane protein stability. We have pursued a strategy for the identification of stabilized variants of the yeast α-factor receptor Ste2p. Because it was not possible to screen directly for mutations providing thermal stabilization, we first isolated a battery of destabilized temperature-sensitive variants, based on loss of signaling function and decreased levels of binding of the fluorescent ligand, and then screened for intragenic second-site suppressors of these phenotypes. The initial screens recovered singly and multiply substituted mutations conferring temperature sensitivity throughout the predicted transmembrane helices of the receptor. All of the singly substituted variants exhibit decreases in cell-surface expression. We then screened randomly mutagenized libraries of clones expressing temperature-sensitive variants for second-site suppressors that restore elevated levels of binding sites for fluorescent ligand. To determine whether any of these were global suppressors, and thus likely stabilizing mutations, they were combined with different temperature-sensitive mutations. Eight of the suppressors exhibited the ability to reverse the defect in ligand binding of multiple temperature-sensitive mutations. Combining certain suppressors into a single allele resulted in levels of suppression greater than that seen with either suppressor alone. Solubilized receptors containing suppressor mutations in the absence of temperature-sensitive mutations exhibit a reduced tendency to aggregate during immobilization on an affinity matrix. Several of the suppressors also exhibit allele-specific behavior indicative of specific intramolecular interactions in the receptor.

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Journal Article | Research Support, N.I.H., Extramural | Research Support, Non-U.S. Gov't

Authors

Zuber J, Danial SA, Connelly SM, Naider F, Dumont ME

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