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Identification of Destabilizing and Stabilizing Mutations of Ste2p, a G Protein-Coupled Receptor in Saccharomyces cerevisiae
journal contribution
posted on 2015-03-10, 00:00 authored by Jeffrey Zuber, Shairy
Azmy Danial, Sara M. Connelly, Fred Naider, Mark E. DumontThe
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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mutagenized librariesSaccharomyces cerevisiaeThe isolationtransmembrane proteinsbinding sitestemperature sensitivityligand bindingaffinity matrixidentificationSolubilized receptorsintramolecular interactionssuppressor mutationstransmembrane helicesmembrane protein stabilityvariants exhibit decreasesSte 2p
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