Evidence for an intermediate conformational state of LacY.

ABSTRACT: 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-?-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.

SUBMITTER: Jiang X 

PROVIDER: S-EPMC3311394 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature