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. 2000 Aug;79(2):747–755. doi: 10.1016/S0006-3495(00)76332-6

Secondary structure components and properties of the melibiose permease from Escherichia coli: a fourier transform infrared spectroscopy analysis.

N Dave 1, A Troullier 1, I Mus-Veteau 1, M Duñach 1, G Leblanc 1, E Padrós 1
PMCID: PMC1300974  PMID: 10920008

Abstract

The structure of the melibiose permease from Escherichia coli has been investigated by Fourier transform infrared spectroscopy, using the purified transporter either in the solubilized state or reconstituted in E. coli lipids. In both instances, the spectra suggest that the permease secondary structure is dominated by alpha-helical components (up to 50%) and contains beta-structure (20%) and additional components assigned to turns, 3(10) helix, and nonordered structures (30%). Two distinct and strong absorption bands are recorded at 1660 and 1653 cm(-1), i.e., in the usual range of absorption of helices of membrane proteins. Moreover, conditions that preserve the transporter functionality (reconstitution in liposomes or solubilization with dodecyl maltoside) make possible the detection of two separate alpha-helical bands of comparable intensity. In contrast, a single intense band, centered at approximately 1656 cm(-1), is recorded from the inactive permease in Triton X-100, or a merged and broader signal is recorded after the solubilized protein is heated in dodecyl maltoside. It is suggested that in the functional permease, distinct signals at 1660 and 1653 cm(-1) arise from two different populations of alpha-helical domains. Furthermore, the sodium- and/or melibiose-induced changes in amide I line shape, and in particular, in the relative amplitudes of the 1660 and 1653 cm(-1) bands, indicate that the secondary structure is modified during the early step of sugar transport. Finally, the observation that approximately 80% of the backbone amide protons can be exchanged suggests high conformational flexibility and/or a large accessibility of the membrane domains to the aqueous solvent.

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Selected References

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  1. Alvarez J., Lee D. C., Baldwin S. A., Chapman D. Fourier transform infrared spectroscopic study of the structure and conformational changes of the human erythrocyte glucose transporter. J Biol Chem. 1987 Mar 15;262(8):3502–3509. [PubMed] [Google Scholar]
  2. Arkin I. T., Russ W. P., Lebendiker M., Schuldiner S. Determining the secondary structure and orientation of EmrE, a multi-drug transporter, indicates a transmembrane four-helix bundle. Biochemistry. 1996 Jun 4;35(22):7233–7238. doi: 10.1021/bi960094i. [DOI] [PubMed] [Google Scholar]
  3. Arrondo J. L., Muga A., Castresana J., Goñi F. M. Quantitative studies of the structure of proteins in solution by Fourier-transform infrared spectroscopy. Prog Biophys Mol Biol. 1993;59(1):23–56. doi: 10.1016/0079-6107(93)90006-6. [DOI] [PubMed] [Google Scholar]
  4. Botfield M. C., Naguchi K., Tsuchiya T., Wilson T. H. Membrane topology of the melibiose carrier of Escherichia coli. J Biol Chem. 1992 Jan 25;267(3):1818–1822. [PubMed] [Google Scholar]
  5. Botfield M. C., Wilson T. H. Mutations that simultaneously alter both sugar and cation specificity in the melibiose carrier of Escherichia coli. J Biol Chem. 1988 Sep 15;263(26):12909–12915. [PubMed] [Google Scholar]
  6. Brandolin G., Doussiere J., Gulik A., Gulik-Krzywicki T., Lauquin G. J., Vignais P. V. Kinetic, binding and ultrastructural properties of the beef heart adenine nucleotide carrier protein after incorporation into phospholipid vesicles. Biochim Biophys Acta. 1980 Oct 3;592(3):592–614. doi: 10.1016/0005-2728(80)90103-6. [DOI] [PubMed] [Google Scholar]
  7. Byler D. M., Susi H. Examination of the secondary structure of proteins by deconvolved FTIR spectra. Biopolymers. 1986 Mar;25(3):469–487. doi: 10.1002/bip.360250307. [DOI] [PubMed] [Google Scholar]
  8. Cabiaux V., Oberg K. A., Pancoska P., Walz T., Agre P., Engel A. Secondary structures comparison of aquaporin-1 and bacteriorhodopsin: a Fourier transform infrared spectroscopy study of two-dimensional membrane crystals. Biophys J. 1997 Jul;73(1):406–417. doi: 10.1016/S0006-3495(97)78080-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chin J. J., Jung E. K., Chen V., Jung C. Y. Structural basis of human erythrocyte glucose transporter function in proteoliposome vesicles: circular dichroism measurements. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4113–4116. doi: 10.1073/pnas.84.12.4113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chin J. J., Jung E. K., Jung C. Y. Structural basis of human erythrocyte glucose transporter function in reconstituted vesicles. J Biol Chem. 1986 Jun 5;261(16):7101–7104. [PubMed] [Google Scholar]
  11. Cladera J., Sabés M., Padrós E. Fourier transform infrared analysis of bacteriorhodopsin secondary structure. Biochemistry. 1992 Dec 15;31(49):12363–12368. doi: 10.1021/bi00164a010. [DOI] [PubMed] [Google Scholar]
  12. Clark A. H., Saunderson D. H., Suggett A. Infrared and laser-Raman spectroscopic studies of thermally-induced globular protein gels. Int J Pept Protein Res. 1981 Mar;17(3):353–364. doi: 10.1111/j.1399-3011.1981.tb02002.x. [DOI] [PubMed] [Google Scholar]
  13. Cordat E., Mus-Veteau I., Leblanc G. Structural studies of the melibiose permease of Escherichia coli by fluorescence resonance energy transfer. II. Identification of the tryptophan residues acting as energy donors. J Biol Chem. 1998 Dec 11;273(50):33198–33202. doi: 10.1074/jbc.273.50.33198. [DOI] [PubMed] [Google Scholar]
  14. Damiano-Forano E., Bassilana M., Leblanc G. Sugar binding properties of the melibiose permease in Escherichia coli membrane vesicles. Effects of Na+ and H+ concentrations. J Biol Chem. 1986 May 25;261(15):6893–6899. [PubMed] [Google Scholar]
  15. Fabian H., Naumann D., Misselwitz R., Ristau O., Gerlach D., Welfle H. Secondary structure of streptokinase in aqueous solution: a Fourier transform infrared spectroscopic study. Biochemistry. 1992 Jul 21;31(28):6532–6538. doi: 10.1021/bi00143a024. [DOI] [PubMed] [Google Scholar]
  16. Goormaghtigh E., Cabiaux V., Ruysschaert J. M. Determination of soluble and membrane protein structure by Fourier transform infrared spectroscopy. I. Assignments and model compounds. Subcell Biochem. 1994;23:329–362. doi: 10.1007/978-1-4615-1863-1_8. [DOI] [PubMed] [Google Scholar]
  17. Gwizdek C., Leblanc G., Bassilana M. Proteolytic mapping and substrate protection of the Escherichia coli melibiose permease. Biochemistry. 1997 Jul 15;36(28):8522–8529. doi: 10.1021/bi970312n. [DOI] [PubMed] [Google Scholar]
  18. Hama H., Wilson T. H. Cation-coupling in chimeric melibiose carriers derived from Escherichia coli and Klebsiella pneumoniae. The amino-terminal portion is crucial for Na+ recognition in melibiose transport. J Biol Chem. 1993 May 15;268(14):10060–10065. [PubMed] [Google Scholar]
  19. Holloway P. W., Mantsch H. H. Structure of cytochrome b5 in solution by Fourier-transform infrared spectroscopy. Biochemistry. 1989 Feb 7;28(3):931–935. doi: 10.1021/bi00429a002. [DOI] [PubMed] [Google Scholar]
  20. Jackson M., Mantsch H. H. The use and misuse of FTIR spectroscopy in the determination of protein structure. Crit Rev Biochem Mol Biol. 1995;30(2):95–120. doi: 10.3109/10409239509085140. [DOI] [PubMed] [Google Scholar]
  21. Krimm S., Dwivedi A. M. Infrared spectrum of the purple membrane: clue to a proton conduction mechanism? Science. 1982 Apr 23;216(4544):407–408. doi: 10.1126/science.6280277. [DOI] [PubMed] [Google Scholar]
  22. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  23. Maehrel C., Cordat E., Mus-Veteau I., Leblanc G. Structural studies of the melibiose permease of Escherichia coli by fluorescence resonance energy transfer. I. Evidence for ion-induced conformational change. J Biol Chem. 1998 Dec 11;273(50):33192–33197. doi: 10.1074/jbc.273.50.33192. [DOI] [PubMed] [Google Scholar]
  24. Mitra A. K., van Hoek A. N., Wiener M. C., Verkman A. S., Yeager M. The CHIP28 water channel visualized in ice by electron crystallography. Nat Struct Biol. 1995 Sep;2(9):726–729. doi: 10.1038/nsb0995-726. [DOI] [PubMed] [Google Scholar]
  25. Mus-Veteau I., Leblanc G. Melibiose permease of Escherichia coli: structural organization of cosubstrate binding sites as deduced from tryptophan fluorescence analyses. Biochemistry. 1996 Sep 17;35(37):12053–12060. doi: 10.1021/bi961372g. [DOI] [PubMed] [Google Scholar]
  26. Mus-Veteau I., Pourcher T., Leblanc G. Melibiose permease of Escherichia coli: substrate-induced conformational changes monitored by tryptophan fluorescence spectroscopy. Biochemistry. 1995 May 23;34(20):6775–6783. doi: 10.1021/bi00020a024. [DOI] [PubMed] [Google Scholar]
  27. Patzlaff J. S., Moeller J. A., Barry B. A., Brooker R. J. Fourier transform infrared analysis of purified lactose permease: a monodisperse lactose permease preparation is stably folded, alpha-helical, and highly accessible to deuterium exchange. Biochemistry. 1998 Nov 3;37(44):15363–15375. doi: 10.1021/bi981142x. [DOI] [PubMed] [Google Scholar]
  28. Poolman B., Konings W. N. Secondary solute transport in bacteria. Biochim Biophys Acta. 1993 Nov 2;1183(1):5–39. doi: 10.1016/0005-2728(93)90003-x. [DOI] [PubMed] [Google Scholar]
  29. Pourcher T., Bassilana M., Sarkar H. K., Kaback H. R., Leblanc G. The melibiose/Na+ symporter of Escherichia coli: kinetic and molecular properties. Philos Trans R Soc Lond B Biol Sci. 1990 Jan 30;326(1236):411–423. doi: 10.1098/rstb.1990.0021. [DOI] [PubMed] [Google Scholar]
  30. Pourcher T., Bibi E., Kaback H. R., Leblanc G. Membrane topology of the melibiose permease of Escherichia coli studied by melB-phoA fusion analysis. Biochemistry. 1996 Apr 2;35(13):4161–4168. doi: 10.1021/bi9527496. [DOI] [PubMed] [Google Scholar]
  31. Pourcher T., Leclercq S., Brandolin G., Leblanc G. Melibiose permease of Escherichia coli: large scale purification and evidence that H+, Na+, and Li+ sugar symport is catalyzed by a single polypeptide. Biochemistry. 1995 Apr 4;34(13):4412–4420. doi: 10.1021/bi00013a033. [DOI] [PubMed] [Google Scholar]
  32. Pourcher T., Zani M. L., Leblanc G. Mutagenesis of acidic residues in putative membrane-spanning segments of the melibiose permease of Escherichia coli. I. Effect on Na(+)-dependent transport and binding properties. J Biol Chem. 1993 Feb 15;268(5):3209–3215. [PubMed] [Google Scholar]
  33. Prestrelski S. J., Tedeschi N., Arakawa T., Carpenter J. F. Dehydration-induced conformational transitions in proteins and their inhibition by stabilizers. Biophys J. 1993 Aug;65(2):661–671. doi: 10.1016/S0006-3495(93)81120-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Reizer J., Reizer A., Saier M. H., Jr A functional superfamily of sodium/solute symporters. Biochim Biophys Acta. 1994 Jun 29;1197(2):133–166. doi: 10.1016/0304-4157(94)90003-5. [DOI] [PubMed] [Google Scholar]
  35. Rigaud J. L., Paternostre M. T., Bluzat A. Mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents. 2. Incorporation of the light-driven proton pump bacteriorhodopsin. Biochemistry. 1988 Apr 19;27(8):2677–2688. doi: 10.1021/bi00408a007. [DOI] [PubMed] [Google Scholar]
  36. Surewicz W. K., Mantsch H. H., Chapman D. Determination of protein secondary structure by Fourier transform infrared spectroscopy: a critical assessment. Biochemistry. 1993 Jan 19;32(2):389–394. doi: 10.1021/bi00053a001. [DOI] [PubMed] [Google Scholar]
  37. Surewicz W. K., Mantsch H. H. New insight into protein secondary structure from resolution-enhanced infrared spectra. Biochim Biophys Acta. 1988 Jan 29;952(2):115–130. doi: 10.1016/0167-4838(88)90107-0. [DOI] [PubMed] [Google Scholar]
  38. Tatulian S. A., Biltonen R. L., Tamm L. K. Structural changes in a secretory phospholipase A2 induced by membrane binding: a clue to interfacial activation? J Mol Biol. 1997 May 23;268(5):809–815. doi: 10.1006/jmbi.1997.1014. [DOI] [PubMed] [Google Scholar]
  39. Tatulian S. A., Cortes D. M., Perozo E. Structural dynamics of the Streptomyces lividans K+ channel (SKC1): secondary structure characterization from FTIR spectroscopy. FEBS Lett. 1998 Feb 20;423(2):205–212. doi: 10.1016/s0014-5793(98)00091-x. [DOI] [PubMed] [Google Scholar]
  40. Torres J., Sepulcre F., Padrós E. Conformational changes in bacteriorhodopsin associated with protein-protein interactions: a functional alpha I-alpha II helix switch? Biochemistry. 1995 Dec 19;34(50):16320–16326. doi: 10.1021/bi00050a012. [DOI] [PubMed] [Google Scholar]
  41. Venyaminov SYu, Kalnin N. N. Quantitative IR spectrophotometry of peptide compounds in water (H2O) solutions. II. Amide absorption bands of polypeptides and fibrous proteins in alpha-, beta-, and random coil conformations. Biopolymers. 1990;30(13-14):1259–1271. doi: 10.1002/bip.360301310. [DOI] [PubMed] [Google Scholar]
  42. Walz T., Typke D., Smith B. L., Agre P., Engel A. Projection map of aquaporin-1 determined by electron crystallography. Nat Struct Biol. 1995 Sep;2(9):730–732. doi: 10.1038/nsb0995-730. [DOI] [PubMed] [Google Scholar]
  43. Wilson D. M., Wilson T. H. Asp-51 and Asp-120 are important for the transport function of the Escherichia coli melibiose carrier. J Bacteriol. 1992 May;174(9):3083–3086. doi: 10.1128/jb.174.9.3083-3086.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wilson D. M., Wilson T. H. Transport properties of Asp-51-->Glu and Asp-120-->Glu mutants of the melibiose carrier of Escherichia coli. Biochim Biophys Acta. 1994 Mar 23;1190(2):225–230. doi: 10.1016/0005-2736(94)90078-7. [DOI] [PubMed] [Google Scholar]
  45. Yazyu H., Shiota-Niiya S., Shimamoto T., Kanazawa H., Futai M., Tsuchiya T. Nucleotide sequence of the melB gene and characteristics of deduced amino acid sequence of the melibiose carrier in Escherichia coli. J Biol Chem. 1984 Apr 10;259(7):4320–4326. [PubMed] [Google Scholar]
  46. de Jongh H. H., Goormaghtigh E., Ruysschaert J. M. The different molar absorptivities of the secondary structure types in the amide I region: an attenuated total reflection infrared study on globular proteins. Anal Biochem. 1996 Nov 1;242(1):95–103. doi: 10.1006/abio.1996.0434. [DOI] [PubMed] [Google Scholar]
  47. le Coutre J., Kaback H. R., Patel C. K., Heginbotham L., Miller C. Fourier transform infrared spectroscopy reveals a rigid alpha-helical assembly for the tetrameric Streptomyces lividans K+ channel. Proc Natl Acad Sci U S A. 1998 May 26;95(11):6114–6117. doi: 10.1073/pnas.95.11.6114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. le Coutre J., Narasimhan L. R., Patel C. K., Kaback H. R. The lipid bilayer determines helical tilt angle and function in lactose permease of Escherichia coli. Proc Natl Acad Sci U S A. 1997 Sep 16;94(19):10167–10171. doi: 10.1073/pnas.94.19.10167. [DOI] [PMC free article] [PubMed] [Google Scholar]

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