A mutation in the lactose permease of Escherichia coli that decreases conformational flexibility and increases protein stability.
Lactose permease with Cys154 --> Gly (helix V) binds substrate with high affinity but catalyzes little or no transport. The purified, detergent-solubilized mutant protein exhibits much greater thermal stability than the wild type and little tendency to aggregate. Stabilization is also observed in vivo with an unstable mutant that is expressed at significantly higher levels when the Cys154 --> Gly mutation is introduced. In addition, ligand-induced conformational changes are markedly reduced or abolished by the Cys154 --> Gly mutation: (i) Although the fluorescence of purified single Trp33 (helix I) permease is enhanced by ligand binding, introduction of the Cys154 --> Gly mutation abolishes the effect. (ii) The rate of 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) labeling of permease with a single Cys residue in place of Val331 (helix X) is increased in the presence of ligand but reduced when the Cys154 --> Gly mutation is present. (iii) Fluorescence emission intensity of MIANS-labeled single Cys331 permease is enhanced and blue shifted in the Cys154 --> Gly mutant background, indicating that the latter mutation causes position 331 to become exposed to a less polar environment. The results indicate that the Cys154 --> Gly mutation causes a more compact structure and decreased conformational flexibility, an alteration that specifically blocks the structural changes necessary for substrate translocation with little or no effect on ligand binding.
Smirnova
Irina N
IN
Howard Hughes Medical Institute, Department of Physiology and Microbiology, University of California, Los Angeles, California 90095-1662, USA.
Kaback
H Ronald
HR
D019943:Amino Acid Substitution / Q000235:genetics*; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D005998:Glycine / Q000235:genetics; D006358:Hot Temperature; D008024:Ligands; D008320:Maltose / Q000031:analogs & derivatives* / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D016254:Mutagenesis, Insertional; D011487:Protein Conformation; D021381:Protein Transport / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D027981:Symporters; D013816:Thermodynamics
D000817:Anilino Naphthalenesulfonates; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008024:Ligands; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C109906:n-lauryl beta-d-maltopyranoside; D008320:Maltose; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; C021499:lactose permease; D003545:Cysteine; D005998:Glycine