Mohindar S. Poonian

Site-directed mutagenesis of cys148 in the lac carrier protein of Escherichia coli

The lac y gene of Escherichia coli which encodes the lac carrier protein has been modified by oligonucleotide-directed, site-specific mutagenesis such that cys148 is converted to a glycine residue. Cells bearing the mutated lac y gene exhibit initial rates of lactose transport that are about 4-fold lower than cells bearing the wild type gene on a recombinant plasmid. Furthermore, transport activity is less sensitive to inactivation by N-ethylmaleimide, and strikingly, galactosyl 1-thio-beta-D-galactopyranoside affords no protection against inactivation. The findings suggest that although cys148 is essential for substrate protection against sulfhydryl inactivation, it is not obligatory for lactose: proton symport and that another sulfhydryl group elsewhere within the lac carrier protein may be required for full activity.

Displacement of [3H] diazepam binding in rat brain by dipyridamole and by 1-methylisoguanosine, a marine natural product with muscle relaxant activity

1-Methylisoguanosine, a marine natural product isolated from the sponge Tedania digitata, and a number of closely-related synthetic analogues have been tested for their ability to displace [3H] diazepam binding to rat brain membranes. Of all the purines yet tested, the natural product was the most active, being some ten-fold better than 2′-deoxyguanosine, the most potent purine so far reported in the literature (7). Other analogues in which only the ribose moiety was altered displayed very similar activity, whereas alteration of the 1-methylisoguanosine base markedly reduced their ability to displace bound diazepam. Of a number of drugs known to interfere with purinergic systems, the adenosine uptake blocker dipyridamole was shown to be relatively potent as a diazepam displacer, with an IC50 of about 300 nM.

cys154 Is important for lac permease activity in Escherichia coli

The lac Y gene of Escherichia coli which encodes the lac permease has been modified by oligonucleotide-directed, site-specific mutagenesis such that cys154 is replaced with either gly or ser. Permease with gly in place of cys154 exhibits essentially no transport activity, while substitution of cys154 with ser also causes marked, though less complete loss of activity. The findings suggest that cys154 plays an important role in lactose:H+ symport.

[17] Oligonucleotide-directed site-specific mutagenesis of the 1ac permease of Escherichia coli

Publisher Summary This chapter illustrates that with the recent advent of site-directed mutagenesis, the goal of determining the role of amino acid residues or sequences in the function of a particular protein or enzyme, can be realized. The chapter describes an application of the technique to the lac permease of Escherichia coli. The lac permease is an intrinsic membrane protein, encoded by the lacY gene, that catalyzes symport (cotransport) of β-galactosides with protons. The lacY gene has been cloned and sequenced, and the permease has been purified to homogeneity in a completely functional state. A putative secondary structure model has been proposed based on the circular dichroic spectrum of the purified protein and on the sequential hydropathic character of the amino acid sequence. Although chemical modification of specific amino acid residues in a protein can provide important information, there are drawbacks to this approach. For this reason, oligonucleotide-directed, sitespecific mutageneses to study the structure and function of the lac permease have been utilized.