Dynamics of lactose permease of Escherichia coli determined by site-directed chemical labeling and fluorescence spectroscopy.
Mutants with a single Cys residue in place of Phe27, Pro28, Phe29, Phe30, or Pro31 at the periplasmic end of putative transmembrane helix I were used to study the interaction of lactose permease with ligand by site-directed chemical modification or fluorescence spectroscopy. With permease embedded in the native membrane, mutant Phe27-->Cys or Phe28-->Cys is readily labeled with [14C]-N-ethylmaleimide (NEM), while mutant Phe29-->Cys, Phe30-->Cys, or Phe31-->Cys reacts less effectively. beta,D-Galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG) has little or no effect on the reactivity of Phe27-->Cys, Phe29-->Cys, or Phe30-->Cys permease. Remarkably, however, Pro31-->Cys permease which is essentially unreactive in the absence of ligand becomes highly reactive in the presence of TDG. Ligand also enhances the NEM reactivity of the mutant with Cys in place of Pro28 which is presumably on the same face of helix I as position 31. The five single-Cys mutants which also contain a biotin acceptor domain in the middle cytoplasmic loop were purified by monomeric avidin-affinity chromatography in dodecyl beta,D-maltoside and subjected to site-directed fluorescence spectroscopy. Mutants Phe27-->Cys, Phe29-->Cys, and Phe30-->Cys react rapidly with 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS), and reactivity is not altered in the presence of TDG. In striking contrast, mutants Pro28-->Cys and Pro31-->Cys react extremely slowly with MIANS in the absent of ligand, and TDG dramatically enhances reactivity.(ABSTRACT TRUNCATED AT 250 WORDS)
Wu
J
J
Howard Hughes Medical Institute, Department of Physiology and Microbiology, University of California, Los Angeles 90095-1662, USA.
Frillingos
S
S
Kaback
H R
HR
D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates; D002240:Carbohydrate Sequence; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide / Q000378:metabolism; D005456:Fluorescent Dyes; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000096:biosynthesis / Q000737:chemistry*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011721:Pyrenes; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents; D027981:Symporters; D013862:Thiogalactosides
D000817:Anilino Naphthalenesulfonates; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011721:Pyrenes; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; D027981:Symporters; D013862:Thiogalactosides; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; C031803:thiodigalactoside; C021499:lactose permease; C030984:pyrene; D003545:Cysteine; D005033:Ethylmaleimide