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. 1999 May;76(5):2702–2710. doi: 10.1016/S0006-3495(99)77422-9

Azide reduces the hydrophobic barrier of the bacteriorhodopsin proton channel.

H J Steinhoff 1, M Pfeiffer 1, T Rink 1, O Burlon 1, M Kurz 1, J Riesle 1, E Heuberger 1, K Gerwert 1, D Oesterhelt 1
PMCID: PMC1300239  PMID: 10233084

Abstract

The sensitivity of a nitroxide spin label to the polarity of its environment has been used to estimate the hydrophobic barrier of the proton channel of the transmembrane proton pump bacteriorhodopsin. By means of site-specific mutagenesis, single cysteine residues were introduced at 10 positions located at the protein surface, in the protein interior, and along the proton pathway. After reaction with a methanethiosulfonate spin label, the principle values of the hyperfine tensor A and the g-tensor were determined from electron paramagnetic resonance spectra measured at 170 K. The shape of the hydrophobic barrier of the proton channel is characterized in terms of a polarity index, DeltaA, determined from the variation of the hyperfine coupling constant Azz. The maximum of the hydrophobic barrier is found to be close to the retinal chromophore in the proton uptake pathway. The effect of the asymmetric distribution of charged and polar residues in the proton release and uptake pathways is clearly reflected in the behavior of the hydrophobic barrier. The presence of azide reduces the barrier height of both the cytoplasmic and extracellular channels. This finding supports the view of azide and other weakly acidic anions as catalysts for the formation of hydrogen-bonded networks in proton pathways of proteins.

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Selected References

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  1. Altenbach C., Greenhalgh D. A., Khorana H. G., Hubbell W. L. A collision gradient method to determine the immersion depth of nitroxides in lipid bilayers: application to spin-labeled mutants of bacteriorhodopsin. Proc Natl Acad Sci U S A. 1994 Mar 1;91(5):1667–1671. doi: 10.1073/pnas.91.5.1667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Altenbach C., Marti T., Khorana H. G., Hubbell W. L. Transmembrane protein structure: spin labeling of bacteriorhodopsin mutants. Science. 1990 Jun 1;248(4959):1088–1092. doi: 10.1126/science.2160734. [DOI] [PubMed] [Google Scholar]
  3. Bashford D., Gerwert K. Electrostatic calculations of the pKa values of ionizable groups in bacteriorhodopsin. J Mol Biol. 1992 Mar 20;224(2):473–486. doi: 10.1016/0022-2836(92)91009-e. [DOI] [PubMed] [Google Scholar]
  4. Brown L. S., Váró G., Needleman R., Lanyi J. K. Functional significance of a protein conformation change at the cytoplasmic end of helix F during the bacteriorhodopsin photocycle. Biophys J. 1995 Nov;69(5):2103–2111. doi: 10.1016/S0006-3495(95)80081-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cao Y., Váró G., Chang M., Ni B. F., Needleman R., Lanyi J. K. Water is required for proton transfer from aspartate-96 to the bacteriorhodopsin Schiff base. Biochemistry. 1991 Nov 12;30(45):10972–10979. doi: 10.1021/bi00109a023. [DOI] [PubMed] [Google Scholar]
  6. Earle K. A., Moscicki J. K., Ge M., Budil D. E., Freed J. H. 250-GHz electron spin resonance studies of polarity gradients along the aliphatic chains in phospholipid membranes. Biophys J. 1994 Apr;66(4):1213–1221. doi: 10.1016/S0006-3495(94)80905-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Essen L., Siegert R., Lehmann W. D., Oesterhelt D. Lipid patches in membrane protein oligomers: crystal structure of the bacteriorhodopsin-lipid complex. Proc Natl Acad Sci U S A. 1998 Sep 29;95(20):11673–11678. doi: 10.1073/pnas.95.20.11673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Farahbakhsh Z. T., Altenbach C., Hubbell W. L. Spin labeled cysteines as sensors for protein-lipid interaction and conformation in rhodopsin. Photochem Photobiol. 1992 Dec;56(6):1019–1033. doi: 10.1111/j.1751-1097.1992.tb09725.x. [DOI] [PubMed] [Google Scholar]
  9. Ferrando E., Schweiger U., Oesterhelt D. Homologous bacterio-opsin-encoding gene expression via site-specific vector integration. Gene. 1993 Mar 15;125(1):41–47. doi: 10.1016/0378-1119(93)90743-m. [DOI] [PubMed] [Google Scholar]
  10. Ganea C., Tittor J., Bamberg E., Oesterhelt D. Chloride- and pH-dependent proton transport by BR mutant D85N. Biochim Biophys Acta. 1998 Jan 5;1368(1):84–96. doi: 10.1016/s0005-2736(97)00173-9. [DOI] [PubMed] [Google Scholar]
  11. Griffith O. H., Dehlinger P. J., Van S. P. Shape of the hydrophobic barrier of phospholipid bilayers (evidence for water penetration in biological membranes). J Membr Biol. 1974;15(2):159–192. doi: 10.1007/BF01870086. [DOI] [PubMed] [Google Scholar]
  12. Grigorieff N., Ceska T. A., Downing K. H., Baldwin J. M., Henderson R. Electron-crystallographic refinement of the structure of bacteriorhodopsin. J Mol Biol. 1996 Jun 14;259(3):393–421. doi: 10.1006/jmbi.1996.0328. [DOI] [PubMed] [Google Scholar]
  13. Hatanaka M., Kandori H., Maeda A. Localization and orientation of functional water molecules in bacteriorhodopsin as revealed by polarized Fourier transform infrared spectroscopy. Biophys J. 1997 Aug;73(2):1001–1006. doi: 10.1016/S0006-3495(97)78133-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hessling B., Souvignier G., Gerwert K. A model-independent approach to assigning bacteriorhodopsin's intramolecular reactions to photocycle intermediates. Biophys J. 1993 Nov;65(5):1929–1941. doi: 10.1016/S0006-3495(93)81264-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hubbell W. L., Mchaourab H. S., Altenbach C., Lietzow M. A. Watching proteins move using site-directed spin labeling. Structure. 1996 Jul 15;4(7):779–783. doi: 10.1016/s0969-2126(96)00085-8. [DOI] [PubMed] [Google Scholar]
  16. Kimura Y., Vassylyev D. G., Miyazawa A., Kidera A., Matsushima M., Mitsuoka K., Murata K., Hirai T., Fujiyoshi Y. Surface of bacteriorhodopsin revealed by high-resolution electron crystallography. Nature. 1997 Sep 11;389(6647):206–211. doi: 10.1038/38323. [DOI] [PubMed] [Google Scholar]
  17. Koch M. H., Dencher N. A., Oesterhelt D., Plöhn H. J., Rapp G., Büldt G. Time-resolved X-ray diffraction study of structural changes associated with the photocycle of bacteriorhodopsin. EMBO J. 1991 Mar;10(3):521–526. doi: 10.1002/j.1460-2075.1991.tb07978.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lam W. L., Doolittle W. F. Shuttle vectors for the archaebacterium Halobacterium volcanii. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5478–5482. doi: 10.1073/pnas.86.14.5478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lanyi J. K. Proton translocation mechanism and energetics in the light-driven pump bacteriorhodopsin. Biochim Biophys Acta. 1993 Dec 7;1183(2):241–261. doi: 10.1016/0005-2728(93)90226-6. [DOI] [PubMed] [Google Scholar]
  20. Le Coutre J., Tittor J., Oesterhelt D., Gerwert K. Experimental evidence for hydrogen-bonded network proton transfer in bacteriorhodopsin shown by Fourier-transform infrared spectroscopy using azide as catalyst. Proc Natl Acad Sci U S A. 1995 May 23;92(11):4962–4966. doi: 10.1073/pnas.92.11.4962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Luecke H., Richter H. T., Lanyi J. K. Proton transfer pathways in bacteriorhodopsin at 2.3 angstrom resolution. Science. 1998 Jun 19;280(5371):1934–1937. doi: 10.1126/science.280.5371.1934. [DOI] [PubMed] [Google Scholar]
  22. Mchaourab H. S., Lietzow M. A., Hideg K., Hubbell W. L. Motion of spin-labeled side chains in T4 lysozyme. Correlation with protein structure and dynamics. Biochemistry. 1996 Jun 18;35(24):7692–7704. doi: 10.1021/bi960482k. [DOI] [PubMed] [Google Scholar]
  23. Oesterhelt D., Meentzen M., Schuhmann L. Reversible dissociation of the purple complex in bacteriorhodopsin and identification of 13-cis and all-trans-retinal as its chromophores. Eur J Biochem. 1973 Dec 17;40(2):453–463. doi: 10.1111/j.1432-1033.1973.tb03214.x. [DOI] [PubMed] [Google Scholar]
  24. Oesterhelt D., Stoeckenius W. Isolation of the cell membrane of Halobacterium halobium and its fractionation into red and purple membrane. Methods Enzymol. 1974;31:667–678. doi: 10.1016/0076-6879(74)31072-5. [DOI] [PubMed] [Google Scholar]
  25. Oesterhelt D. The structure and mechanism of the family of retinal proteins from halophilic archaea. Curr Opin Struct Biol. 1998 Aug;8(4):489–500. doi: 10.1016/s0959-440x(98)80128-0. [DOI] [PubMed] [Google Scholar]
  26. Pebay-Peyroula E., Rummel G., Rosenbusch J. P., Landau E. M. X-ray structure of bacteriorhodopsin at 2.5 angstroms from microcrystals grown in lipidic cubic phases. Science. 1997 Sep 12;277(5332):1676–1681. doi: 10.1126/science.277.5332.1676. [DOI] [PubMed] [Google Scholar]
  27. Pfeiffer M., Rink T., Gerwert K., Oesterhelt D., Steinhoff H. J. Site-directed spin-labeling reveals the orientation of the amino acid side-chains in the E-F loop of bacteriorhodopsin. J Mol Biol. 1999 Mar 19;287(1):163–171. doi: 10.1006/jmbi.1998.2593. [DOI] [PubMed] [Google Scholar]
  28. Rammelsberg R., Huhn G., Lübben M., Gerwert K. Bacteriorhodopsin's intramolecular proton-release pathway consists of a hydrogen-bonded network. Biochemistry. 1998 Apr 7;37(14):5001–5009. doi: 10.1021/bi971701k. [DOI] [PubMed] [Google Scholar]
  29. Riesle J., Oesterhelt D., Dencher N. A., Heberle J. D38 is an essential part of the proton translocation pathway in bacteriorhodopsin. Biochemistry. 1996 May 28;35(21):6635–6643. doi: 10.1021/bi9600456. [DOI] [PubMed] [Google Scholar]
  30. Rink T., Riesle J., Oesterhelt D., Gerwert K., Steinhoff H. J. Spin-labeling studies of the conformational changes in the vicinity of D36, D38, T46, and E161 of bacteriorhodopsin during the photocycle. Biophys J. 1997 Aug;73(2):983–993. doi: 10.1016/S0006-3495(97)78131-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schulte A., Bradley L., 2nd High-pressure near-infrared Raman spectroscopy of bacteriorhodopsin light to dark adaptation. Biophys J. 1995 Oct;69(4):1554–1562. doi: 10.1016/S0006-3495(95)80027-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Steinhoff H. J. A simple method for determination of rotational correlation times and separation of rotational and polarity effects from EPR spectra of spin-labeled biomolecules in a wide correlation time range. J Biochem Biophys Methods. 1988 Dec;17(4):237–247. doi: 10.1016/0165-022x(88)90047-4. [DOI] [PubMed] [Google Scholar]
  33. Steinhoff H. J., Hubbell W. L. Calculation of electron paramagnetic resonance spectra from Brownian dynamics trajectories: application to nitroxide side chains in proteins. Biophys J. 1996 Oct;71(4):2201–2212. doi: 10.1016/S0006-3495(96)79421-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Steinhoff H. J., Mollaaghababa R., Altenbach C., Hideg K., Krebs M., Khorana H. G., Hubbell W. L. Time-resolved detection of structural changes during the photocycle of spin-labeled bacteriorhodopsin. Science. 1994 Oct 7;266(5182):105–107. doi: 10.1126/science.7939627. [DOI] [PubMed] [Google Scholar]
  35. Subramaniam S., Faruqi A. R., Oesterhelt D., Henderson R. Electron diffraction studies of light-induced conformational changes in the Leu-93 --> Ala bacteriorhodopsin mutant. Proc Natl Acad Sci U S A. 1997 Mar 4;94(5):1767–1772. doi: 10.1073/pnas.94.5.1767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Subramaniam S., Gerstein M., Oesterhelt D., Henderson R. Electron diffraction analysis of structural changes in the photocycle of bacteriorhodopsin. EMBO J. 1993 Jan;12(1):1–8. doi: 10.1002/j.1460-2075.1993.tb05625.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tittor J., Soell C., Oesterhelt D., Butt H. J., Bamberg E. A defective proton pump, point-mutated bacteriorhodopsin Asp96----Asn is fully reactivated by azide. EMBO J. 1989 Nov;8(11):3477–3482. doi: 10.1002/j.1460-2075.1989.tb08512.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Váró G., Lanyi J. K. Thermodynamics and energy coupling in the bacteriorhodopsin photocycle. Biochemistry. 1991 May 21;30(20):5016–5022. doi: 10.1021/bi00234a025. [DOI] [PubMed] [Google Scholar]
  39. Weik M., Zaccai G., Dencher N. A., Oesterhelt D., Hauss T. Structure and hydration of the M-state of the bacteriorhodopsin mutant D96N studied by neutron diffraction. J Mol Biol. 1998 Jan 30;275(4):625–634. doi: 10.1006/jmbi.1997.1488. [DOI] [PubMed] [Google Scholar]
  40. Wikström M. Proton translocation by bacteriorhodopsin and heme-copper oxidases. Curr Opin Struct Biol. 1998 Aug;8(4):480–488. doi: 10.1016/s0959-440x(98)80127-9. [DOI] [PubMed] [Google Scholar]

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