Abstract
In order to understand the unusual heat resistance of LamB protein (the outer membrane component of the maltose transport system in Escherichia coli and its receptor for bacteriophage lambda), we investigated the role of its 2 cysteinyl residues. Our studies show that Cys22 and Cys38 form an intrasubunit disulfide bond which contributes to the heat stability of the LamB protein trimer. Physical evidence for the disulfide was obtained by using site-directed mutagenesis to convert Asn36 to Met, which allowed cyanogen bromide cleavage between the 2 cysteines. Upon reduction one of the N36M fragments migrated as two pieces, resolved by two-dimensional polyacrylamide gel electrophoresis. Other mutagenized LamB proteins, in which 1 or both Cys residues were converted to Ser, exhibited a sharp loss of thermal stability. In contrast to wild-type LamB protein trimer, which does not dissociate to monomers even after 60 min at 100 degrees C, only 10-15% of the mutant LamB proteins remain trimeric after boiling 10 min. The disulfide bond in LamB protein is not required for its transport function, since both mutagenized LamB protein and N-ethylmaleimide-labeled LamB protein exhibit normal uptake of sugars in proteoliposomes. Finally, the disulfide bond must not be between subunits of the LamB trimer since reversible dissociation of trimer is achieved by low pH or denaturants in the absence of reducing agent.
Highlights
Met, which allowed cyanogen bromide cleavage between the 2 cysteines
Physical Evidence for a Disulfide Bond in LamB ProteinTo determine whether the cysteines form a disulfide bond in
Using site-directed mutagenesis to place a Met between the 2 Cys residues of LamB protein allowed us to demonstrate the presence of an non-essential disulfide bond
Summary
$ To whom correspondence should be addressed. the LamB protein trimer. It is resistant to denaturation by sodium dodecyl sulfate (Nakae,1979)and difficult to denature with urea or guanidine hydrochloride (Kuhl, 1980; Page and Rosenbusch, 1986). In this work we investigated the conditions promoting dissociation of the purified LamB protein trimer, and we used chemical modification and site-directed mutagenesis to study the role of the cysteines. Oligonucleotide-directed Mutagenesis-The lamB gene had been cloned under an IS-3 promoter on the plasmid pTroyl, obtained from C. For overproduction the 1.7-kb Stul fragment of pTroyll was ligated intothe SmaI-digested plasmid pKK223-3, putting theZamB gene under control of the tac promoter. The abbreviations used are: SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; Tricine, Tris(hydroxymethy1)methyl glycine;Napi,sodium phosphate buffer; PME, P-mercaptoethanol; DTT, dithiothreitol; NEM, N-ethylmaleimide; PMSF, phenylmethylsulfonyl fluoride; CNBr, cyanogen bromide
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