Department of Physiology, University of California, Los Angeles, CA 90095, USA.
Proc Natl Acad Sci U S A. 2012 Mar 20;109(12):E698-704. doi: 10.1073/pnas.1201107109. Epub 2012 Feb 21.
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-β-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.
LacY 突变体 Cys154→Gly 表现出与 WT 相同的周质封闭晶体结构，但在膜中是周质开放的。该突变体几乎不能催化运输，但与伪 WT 相比，它以相同的亲和力从膜的两侧结合半乳糖苷，并对定点蛋白酶解具有抗性。定点烷基化也应用于 Cys154→Gly LacY 中的 11 个单-Cys 突变体，在膜外翻小泡中或在十二烷基-β-D-麦芽糖苷（DDM）中溶解和纯化后。与伪 WT 不同的是，Cys154→Gly 突变体在没有糖的情况下，在膜的周质侧快速标记，但在突变体蛋白纯化后标记显著减少。因此，Cys154→Gly LacY 可能在原位优先形成一种能量更高的周质开放构象中间体，但在纯化后折叠成能量更低的周质封闭构象。值得注意的是，支链或新戊二醇麦芽糖苷去污剂稳定了 Cys154→Gly LacY 在膜嵌入形式中。
