Identification of destabilizing and stabilizing mutations of Ste2p, a G protein-coupled receptor in Saccharomyces cerevisiae.
Identification of destabilizing and stabilizing mutations of Ste2p, a G protein-coupled receptor in Saccharomyces cerevisiae.
复制标题
酿酒酵母中 G 蛋白偶联受体 Ste2p 的不稳定和稳定突变的鉴定。
DOI：
10.1021/bi501314t
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发表时间：
2015
期刊：
影响因子：
2.9
通讯作者：
Dumont,MarkE
中科院分区：
文献类型：
--
作者：
Zuber,Jeffrey;Danial,ShairyAzmy;Connelly,SaraM;Naider,Fred;Dumont,MarkE
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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影响因子：
5.7
作者：
Sonoda, Yo;Newstead, Simon;Hu, Nien-Jen;Alguel, Yilmaz;Nji, Emmanuel;Beis, Konstantinos;Yashiro, Shoko;Lee, Chiara;Leung, James;Cameron, Alexander D.;Byrne, Bernadette;Iwata, So;Drew, David
通讯作者：
Drew, David
DOI：
--
发表时间：
2012
期刊：
--
影响因子：
--
作者：
Hui-Lin Wu;Daniel Wacker;Mauro Mileni;V. Katritch;Gye Won Han;Eyal Vardy;Wei Liu;Aaron A. Thompson-Aaron-A.-Th
通讯作者：
Hui-Lin Wu;Daniel Wacker;Mauro Mileni;V. Katritch;Gye Won Han;Eyal Vardy;Wei Liu;Aaron A. Thompson-Aaron-A.-Th
影响因子：
56.9
作者：
KING, K;DOHLMAN, HG;LEFKOWITZ, RJ
通讯作者：
LEFKOWITZ, RJ
哺乳动物生长抑素受体与酵母信息素反应途径的功能耦合
影响因子：
5.3
作者：
L. A. Price;E. M. Kajkowski;J. Hadcock;B. Ozenberger;M. Pausch
通讯作者：
M. Pausch
控制酵母 G 蛋白偶联受体激活和运输的机制。
影响因子：
3.3
作者：
Stefan,CJ;Overton,MC;Blumer,KJ
通讯作者：
Blumer,KJ