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H R Kaback

Publications and source records attributed to H R Kaback.

330 records · Page 19Linked to original sources
PubMedDate not supplied

The lac permease of Escherichia coli: site-directed mutagenesis studies on the mechanism of beta-galactoside/H+ symport.

In this communication, we summarize site-directed mutagenesis studies of the lac permease from Escherichia coli, a prototypic H(+)-coupled active transport protein. We classify mutant permeases by phenotype, and suggest possible roles for some individual residues in the mechanism of H+/lactose symport. Although high-resolution structural information is not presently available, kinetic analysis of the partial reactions catalysed by the mutant permeases, as well as biophysical studies, suggest an evolving model for the mechanism of H+/lactose symport.

Amino Acid Sequence↗
PubMedDate not supplied

Role of glutamate-269 in the lactose permease of Escherichia coli.

Glu-269, which is located on the hydrophilic face of putative helix VIII in the lactose permease of Escherichia coli, has been replaced with Asp, Gln or Cys by oligonucleotide-directed, site specific mutagenesis. Cells expressing Asp-269 permease exhibit no lactose accumulation or lactose-induced H+ translocation, but retain some ability to mediate lactose influx down a concentration gradient at high substrate concentrations. Furthermore, right-side-out membrane vesicles containing Asp-269 permease do not catalyse active lactose transport, facilitated lactose efflux or equilibrium exchange. Remarkably, however, Asp-269 permease accumulates beta, D-galactopyranosyl 1-thio-beta,D-galactopyranoside in a partially uncoupled fashion, whereas no transport of methyl-beta,D-thiogalactopyranoside, sucrose or maltose is detectable. Mutant permeases containing neutral replacements (Gln or Cys) or Glu-269 are completely devoid of activity, although the proteins are present in the membrane at concentrations comparable with wild-type or Asp-269 permease. The observations demonstrate that a carboxylate at position 269 is essential for transport activity, and Glu-269 is important for substrate binding and/or recognition.

Amino Acid Sequence↗
PubMedDate not supplied

In vitro folding of a membrane protein: effect of denaturation and renaturation on substrate binding by the lactose permease of Escherichia coli.

Site-directed mutagenesis and site-directed fluorescence spectroscopy demonstrate that Cys148 interacts hydrophobically with the galactosyl moiety of substrates of the lactose permease of Escherichia coli. By taking advantage of the finding that labelling of single-Cys148 permease with the thiol-specific fluorophore 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) is blocked specifically by substrates of the permease, it is demonstrated that the high-affinity ligand beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG) stabilizes solubilized, purified permease against heat denaturation. Furthermore, TDG protection against MIANS labelling of single-Cys148 permease is abolished by guanidinium hydrochloride. After dialysis of the denaturant, TDG protection against MIANS labelling is recovered, indicating that the permease has been refolded. The conclusion is confirmed and extended by studying site-directed fluorescence of purified single-Cys331 permease, where the emission spectrum of the MIANS-labelled protein is differentially altered by low or high concentrations of TDG. The results demonstrate that both low- and high-affinity binding, as well as ligand-induced conformational changes in the permease, can be denatured reversibly in vitro.

Amino Acid Substitution↗