Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1994 Dec;3(12):2294–2301. doi: 10.1002/pro.5560031214

Ligand-induced conformational changes in the lactose permease of Escherichia coli: evidence for two binding sites.

J Wu 1, S Frillingos 1, J Voss 1, H R Kaback 1
PMCID: PMC2142758  PMID: 7756985

Abstract

By using a lactose permease mutant containing a single Cys residue in place of Val 331 (helix X), conformational changes induced by ligand binding were studied. With right-side-out membrane vesicles containing Val 331-->Cys permease, lactose transport is inactivated by either N-ethylmaleimide (NEM) or 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM). Remarkably, beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG) enhances the rate of inactivation by CPM, a hydrophobic sulfhydryl reagent, whereas NEM inactivation is attenuated by the ligand. Val 331-->Cys permease was then purified and studied in dodecyl-beta,D-maltoside by site-directed fluorescence spectroscopy. The reactivity of Val 331-->Cys permease with 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (MIANS) is not changed over a low range of TDG concentrations (< 0.8 mM), but the fluorescence of the MIANS-labeled protein is quenched in a saturable manner (apparent Kd approximately equal to 0.12 mM) without a change in emission maximum. In contrast, over a higher range of TDG concentrations (1-10 mM), the reactivity of Val 331-->Cys permease with MIANS is enhanced and the emission maximum of MIANS-labeled permease is blue shifted by 3-7 nm. Furthermore, the fluorescence of MIANS-labeled Val 331 -->Cys permease is quenched by both acrylamide and iodide, but the former is considerably more effective. A low concentration of TDG (0.2 mM) does not alter quenching by either compound, whereas a higher concentration of ligand (10 mM) decreases the quenching constant for iodide by about 50% and for acrylamide by about 20%.(ABSTRACT TRUNCATED AT 250 WORDS)

Full Text

The Full Text of this article is available as a PDF (840.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bigelow D. J., Inesi G. Frequency-domain fluorescence spectroscopy resolves the location of maleimide-directed spectroscopic probes within the tertiary structure of the Ca-ATPase of sarcoplasmic reticulum. Biochemistry. 1991 Feb 26;30(8):2113–2125. doi: 10.1021/bi00222a016. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Büchel D. E., Gronenborn B., Müller-Hill B. Sequence of the lactose permease gene. Nature. 1980 Feb 7;283(5747):541–545. doi: 10.1038/283541a0. [DOI] [PubMed] [Google Scholar]
  4. Calamia J., Manoil C. lac permease of Escherichia coli: topology and sequence elements promoting membrane insertion. Proc Natl Acad Sci U S A. 1990 Jul;87(13):4937–4941. doi: 10.1073/pnas.87.13.4937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Consler T. G., Persson B. L., Jung H., Zen K. H., Jung K., Privé G. G., Verner G. E., Kaback H. R. Properties and purification of an active biotinylated lactose permease from Escherichia coli. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):6934–6938. doi: 10.1073/pnas.90.15.6934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Foster D. L., Boublik M., Kaback H. R. Structure of the lac carrier protein of Escherichia coli. J Biol Chem. 1983 Jan 10;258(1):31–34. [PubMed] [Google Scholar]
  7. Gupte S. S., Lane L. K. Reaction of purified (Na,K)-ATPase with the fluorescent sulfhydryl probe 2-(4'-maleimidylanilino)naphthalene 6-sulfonic acid. Characterization and the effects of ligands. J Biol Chem. 1979 Oct 25;254(20):10362–10369. [PubMed] [Google Scholar]
  8. Hattori M., Sakaki Y. Dideoxy sequencing method using denatured plasmid templates. Anal Biochem. 1986 Feb 1;152(2):232–238. doi: 10.1016/0003-2697(86)90403-3. [DOI] [PubMed] [Google Scholar]
  9. Jung K., Jung H., Kaback H. R. Dynamics of lactose permease of Escherichia coli determined by site-directed fluorescence labeling. Biochemistry. 1994 Apr 5;33(13):3980–3985. doi: 10.1021/bi00179a026. [DOI] [PubMed] [Google Scholar]
  10. Jung K., Jung H., Wu J., Privé G. G., Kaback H. R. Use of site-directed fluorescence labeling to study proximity relationships in the lactose permease of Escherichia coli. Biochemistry. 1993 Nov 23;32(46):12273–12278. doi: 10.1021/bi00097a001. [DOI] [PubMed] [Google Scholar]
  11. Kaback H. R., Jung K., Jung H., Wu J., Privé G. G., Zen K. What's new with lactose permease. J Bioenerg Biomembr. 1993 Dec;25(6):627–636. doi: 10.1007/BF00770250. [DOI] [PubMed] [Google Scholar]
  12. Konings W. N., Barnes E. M., Jr, Kaback H. R. Mechanisms of active transport in isolated membrane vesicles. 2. The coupling of reduced phenazine methosulfate to the concentrative uptake of beta-galactosides and amino acids. J Biol Chem. 1971 Oct 10;246(19):5857–5861. [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. Lolkema J. S., Carrasco N., Kaback H. R. Kinetic analysis of lactose exchange in proteoliposomes reconstituted with purified lac permease. Biochemistry. 1991 Feb 5;30(5):1284–1290. doi: 10.1021/bi00219a018. [DOI] [PubMed] [Google Scholar]
  15. Lolkema J. S., Walz D. Binding of p-nitrophenyl alpha-D-galactopyranoside to lac permease of Escherichia coli. Biochemistry. 1990 Dec 25;29(51):11180–11188. doi: 10.1021/bi00503a005. [DOI] [PubMed] [Google Scholar]
  16. Peerce B. E., Wright E. M. Examination of the Na+-induced conformational change of the intestinal brush border sodium/glucose symporter using fluorescent probes. Biochemistry. 1987 Jul 14;26(14):4272–4279. doi: 10.1021/bi00388a014. [DOI] [PubMed] [Google Scholar]
  17. Sahin-Tóth M., Kaback H. R. Cysteine scanning mutagenesis of putative transmembrane helices IX and X in the lactose permease of Escherichia coli. Protein Sci. 1993 Jun;2(6):1024–1033. doi: 10.1002/pro.5560020615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Sahin-Tóth M., Lawrence M. C., Kaback H. R. Properties of permease dimer, a fusion protein containing two lactose permease molecules from Escherichia coli. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5421–5425. doi: 10.1073/pnas.91.12.5421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Short S. A., Kaback H. R., Kohn L. D. Localization of D-lactate dehydrogenase in native and reconstituted Escherichia coli membrane vesicles. J Biol Chem. 1975 Jun 10;250(11):4291–4296. [PubMed] [Google Scholar]
  21. Teather R. M., Bramhall J., Riede I., Wright J. K., Fürst M., Aichele G., Wilhelm U., Overath P. Lactose carrier protein of Escherichia coli. Structure and expression of plasmids carrying the Y gene of the lac operon. Eur J Biochem. 1980;108(1):223–231. doi: 10.1111/j.1432-1033.1980.tb04715.x. [DOI] [PubMed] [Google Scholar]
  22. Viitanen P., Newman M. J., Foster D. L., Wilson T. H., Kaback H. R. Purification, reconstitution, and characterization of the lac permease of Escherichia coli. Methods Enzymol. 1986;125:429–452. doi: 10.1016/s0076-6879(86)25034-x. [DOI] [PubMed] [Google Scholar]
  23. van Iwaarden P. R., Driessen A. J., Lolkema J. S., Kaback H. R., Konings W. N. Exchange, efflux, and substrate binding by cysteine mutants of the lactose permease of Escherichia coli. Biochemistry. 1993 May 25;32(20):5419–5424. doi: 10.1021/bi00071a017. [DOI] [PubMed] [Google Scholar]
  24. van Iwaarden P. R., Pastore J. C., Konings W. N., Kaback H. R. Construction of a functional lactose permease devoid of cysteine residues. Biochemistry. 1991 Oct 8;30(40):9595–9600. doi: 10.1021/bi00104a005. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES