文章基本信息

标题：Evidence for an intermediate conformational state of LacY
本地全文：下载
作者：Xiaoxu Jiang ; Lan Guan ; Yonggang Zhou 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2012
卷号：109
期号：12
页码：E698-E704
DOI：10.1073/pnas.1201107109
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：LacY mutant Cys154 [->] Gly exhibits a periplasmic-closed crystal structure identical to the WT, but is periplasmic-open in the membrane. The mutant hardly catalyzes transport, but binds galactosides from either side of the membrane with the same affinity and is resistant to site-directed proteolysis relative to the pseudo-WT. Site-directed alkylation was also applied to 11 single-Cys mutants in Cys154 [->] Gly LacY in right-side-out membrane vesicles or after solubilization and purification in dodecyl-{beta}-D-maltopyranoside (DDM). Unlike the pseudo-WT, Cys replacements on the periplasmic side of the Cys154 [->] Gly mutant label rapidly in the membrane without sugar, but labeling decreases markedly after the mutant proteins are purified. Thus, Cys154 [->] Gly LacY likely favors a higher-energy intermediate periplasmic-open conformation in situ, but collapses to a lower-energy periplasmic-closed conformation in DDM after purification. Notably, branched-chain or neopentyl glycol maltoside detergents stabilize Cys154 [->] Gly LacY in the membrane-embedded form.
关键词：lactose permease ; membrane proteins ; site-directed alkylation ; symport ; membrane transport