Amino acid substitution in the lactose carrier protein with the use of amber suppressors

Abstract

Five lacY mutants with amber stop codons at known positions were each placed into 12 different suppressor strains. The 60 amino acid substitutions obtained in this manner were tested for growth on lactose-minimal medium plates and for transport of lactose, melibiose, and thiomethylgalactoside. Most of the amino acid substitutions in the regions of the putative loops (between transmembrane a helices) resulted in a reasonable growth rate on lactose with moderate-to-good transport activity. In one strain (glycine substituted for Trp-10), abnormal sugar recognition was found. The substitution of proline for Trp-33 (in the region of the first a helix) showed no activity, while four additional substitutions (lysine, leucine, cysteine, and glutamic acid) showed low activity. Altered sugar specificity was observed when Trp-33 was replaced by serine, glutamine, tyrosine, alanine, histidine, or phenylalanine. It is conduded that Trp-33 may be involved directly or indirectly in sugar recognition.In recent years, it has been possible to study the correlation between structure at the amino acid level and the function of membrane transport proteins. At present, there is a great deal of data available on specific mutants of the lactose carrier of Escherichia coli. More than 150 mutants have been isolated and studied for various aspects of transport. Two general techniques have been used to generate these mutants. The first of these has been the isolation of spontaneous mutants by using a variety of special selection techniques (1-3, 16). The second method is that of site-directed mutagenesis (11,17,22) or cassette mutagenesis (9). Mutants derived from these procedures have been extremely useful in determining amino acid residues which are essential for normal transport function and may be involved in either sugar recognition or coupling of cation movement to sugar transport (for reviews, see references 1 and 22).A useful variation on the point mutagenesis theme involves the use of natural as well as synthetic amber suppressor strains (13,(18)(19)(20)

Fluorescence of native single‐Trp mutants in the lactose permease from Escherichia coli: Structural properties and evidence for a substrate‐induced conformational change

“…Therefore, it appears that occupancy of a high-affinity site in lactose permease causes position 33 to move into a more hydrophobic environment that is less accessible to MIANS, and positions 28 and 3 1 to move into a more hydrophilic environment that is more accessible to the probe. It has been postulated that Trp 33 could be directly or indirectly involved in sugar recognition, based on the finding that substitution of Trp 33 by Ser, Gln, Tyr, Ala, Gly, and Phe leads to higher rates of thiomethylgalactoside transport relative to lactose or melibiose (Huang et al, 1992).…”

Secondary solute transport in bacteria

“…The mutant protein, devoid of all the tryptophan residues, retains more than 70% of the activity of the wild-type protein, indicating that tryptophans have no essential role in the function and structure of LacY. By analyzing various substitutions for Trp-33, this residue has, however, been implicated in sugar recognition [282]. Each of the 14 tyrosine residues of LacY have been replaced with phenylalanine, and the effects of the mutations have been analyzed [283].…”

Predicting Deleterious Amino Acid Substitutions

“…If nonconservative substitutions are predicted to be deleterious, then many substitutions will be predicted to affect phenotype. However, proteins actually contain many positions that have a high degree of plasticity in accommodating amino acid substitutions, as shown in previous mutagenesis studies (Bowie and Sauer 1989;Climie et al 1990;Huang et al 1992;Markiewicz et al 1994). Therefore, experimentally testing all changes deemed nonconservative by a substitution matrix would be time-consuming and wasteful because of this overprediction, especially for large-scale studies such as examination of nonsynonymous SNPs (Lander 1996;Irizarry et al 2000) or in genome-wide random mutagenesis projects (Bentley et al 2000;Chen et al 2000;McCallum et al 2000).…”