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. 2001 Jun;80(6):2856–2866. doi: 10.1016/S0006-3495(01)76252-2

Time-resolved detection of transient movement of helices F and G in doubly spin-labeled bacteriorhodopsin.

N Radzwill 1, K Gerwert 1, H J Steinhoff 1
PMCID: PMC1301470  PMID: 11371459

Abstract

Photo-excited structural changes of the light-driven proton pump bacteriorhodopsin were monitored using double-site-directed spin labeling combined with electron paramagnetic resonance (EPR) spectroscopy. The inter-spin distances between nitroxides attached at residue positions 100 and 226, 101 and 160, and 101 and 168 were determined for the BR initial state and the trapped M photo-intermediate. Distance changes that occur during the photocycle were followed with millisecond time resolution under physiological conditions at 293 K. The kinetic analysis of the EPR data and comparison with the absorbance changes in the visible spectrum reveal an outward movement of helix F during the late M intermediate and a subsequent approach of helix G toward the proton channel. The displacements of the cytoplasmic moieties of these helices amount to 0.1-0.2 nm. We propose that the resulting opening of the proton channel decreases the pK of the proton donor D96 and facilitates proton transfer to the Schiff base during the M-to-N transition.

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Selected References

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  1. Dencher N. A., Dresselhaus D., Zaccai G., Büldt G. Structural changes in bacteriorhodopsin during proton translocation revealed by neutron diffraction. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7876–7879. doi: 10.1073/pnas.86.20.7876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Druckmann S., Friedman N., Lanyi J. K., Needleman R., Ottolenghi M., Sheves M. The back photoreaction of the M intermediate in the photocycle of bacteriorhodopsin: mechanism and evidence for two M species. Photochem Photobiol. 1992;56(6):1041–1047. doi: 10.1111/j.1751-1097.1992.tb09727.x. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Farrens D. L., Altenbach C., Yang K., Hubbell W. L., Khorana H. G. Requirement of rigid-body motion of transmembrane helices for light activation of rhodopsin. Science. 1996 Nov 1;274(5288):768–770. doi: 10.1126/science.274.5288.768. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Haupts U., Tittor J., Oesterhelt D. Closing in on bacteriorhodopsin: progress in understanding the molecule. Annu Rev Biophys Biomol Struct. 1999;28:367–399. doi: 10.1146/annurev.biophys.28.1.367. [DOI] [PubMed] [Google Scholar]
  7. Hessling B., Herbst J., Rammelsberg R., Gerwert K. Fourier transform infrared double-flash experiments resolve bacteriorhodopsin's M1 to M2 transition. Biophys J. 1997 Oct;73(4):2071–2080. doi: 10.1016/S0006-3495(97)78237-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Hubbell W. L., Cafiso D. S., Altenbach C. Identifying conformational changes with site-directed spin labeling. Nat Struct Biol. 2000 Sep;7(9):735–739. doi: 10.1038/78956. [DOI] [PubMed] [Google Scholar]
  10. Hubbell W. L., Gross A., Langen R., Lietzow M. A. Recent advances in site-directed spin labeling of proteins. Curr Opin Struct Biol. 1998 Oct;8(5):649–656. doi: 10.1016/s0959-440x(98)80158-9. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Hustedt E. J., Beth A. H. Nitroxide spin-spin interactions: applications to protein structure and dynamics. Annu Rev Biophys Biomol Struct. 1999;28:129–153. doi: 10.1146/annurev.biophys.28.1.129. [DOI] [PubMed] [Google Scholar]
  13. Kamikubo H., Kataoka M., Váró G., Oka T., Tokunaga F., Needleman R., Lanyi J. K. Structure of the N intermediate of bacteriorhodopsin revealed by x-ray diffraction. Proc Natl Acad Sci U S A. 1996 Feb 20;93(4):1386–1390. doi: 10.1073/pnas.93.4.1386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kensch O., Connolly B. A., Steinhoff H. J., McGregor A., Goody R. S., Restle T. HIV-1 reverse transcriptase-pseudoknot RNA aptamer interaction has a binding affinity in the low picomolar range coupled with high specificity. J Biol Chem. 2000 Jun 16;275(24):18271–18278. doi: 10.1074/jbc.M001309200. [DOI] [PubMed] [Google Scholar]
  15. Krebs M. P., Hauss T., Heyn M. P., RajBhandary U. L., Khorana H. G. Expression of the bacterioopsin gene in Halobacterium halobium using a multicopy plasmid. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):859–863. doi: 10.1073/pnas.88.3.859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Luecke H., Schobert B., Richter H. T., Cartailler J. P., Lanyi J. K. Structural changes in bacteriorhodopsin during ion transport at 2 angstrom resolution. Science. 1999 Oct 8;286(5438):255–261. doi: 10.1126/science.286.5438.255. [DOI] [PubMed] [Google Scholar]
  17. Mchaourab H. S., Oh K. J., Fang C. J., Hubbell W. L. Conformation of T4 lysozyme in solution. Hinge-bending motion and the substrate-induced conformational transition studied by site-directed spin labeling. Biochemistry. 1997 Jan 14;36(2):307–316. doi: 10.1021/bi962114m. [DOI] [PubMed] [Google Scholar]
  18. Mollaaghababa R., Steinhoff H. J., Hubbell W. L., Khorana H. G. Time-resolved site-directed spin-labeling studies of bacteriorhodopsin: loop-specific conformational changes in M. Biochemistry. 2000 Feb 8;39(5):1120–1127. doi: 10.1021/bi991963h. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Oka T., Kamikubo H., Tokunaga F., Lanyi J. K., Needleman R., Kataoka M. Conformational change of helix G in the bacteriorhodopsin photocycle: investigation with heavy atom labeling and x-ray diffraction. Biophys J. 1999 Feb;76(2):1018–1023. doi: 10.1016/S0006-3495(99)77266-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Perozo E., Cortes D. M., Cuello L. G. Structural rearrangements underlying K+-channel activation gating. Science. 1999 Jul 2;285(5424):73–78. doi: 10.1126/science.285.5424.73. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Rink T., Pfeiffer M., Oesterhelt D., Gerwert K., Steinhoff H. J. Unraveling photoexcited conformational changes of bacteriorhodopsin by time resolved electron paramagnetic resonance spectroscopy. Biophys J. 2000 Mar;78(3):1519–1530. doi: 10.1016/S0006-3495(00)76704-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sass H. J., Büldt G., Gessenich R., Hehn D., Neff D., Schlesinger R., Berendzen J., Ormos P. Structural alterations for proton translocation in the M state of wild-type bacteriorhodopsin. Nature. 2000 Aug 10;406(6796):649–653. doi: 10.1038/35020607. [DOI] [PubMed] [Google Scholar]
  26. Sass H. J., Schachowa I. W., Rapp G., Koch M. H., Oesterhelt D., Dencher N. A., Büldt G. The tertiary structural changes in bacteriorhodopsin occur between M states: X-ray diffraction and Fourier transform infrared spectroscopy. EMBO J. 1997 Apr 1;16(7):1484–1491. doi: 10.1093/emboj/16.7.1484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Steinhoff H. J., Radzwill N., Thevis W., Lenz V., Brandenburg D., Antson A., Dodson G., Wollmer A. Determination of interspin distances between spin labels attached to insulin: comparison of electron paramagnetic resonance data with the X-ray structure. Biophys J. 1997 Dec;73(6):3287–3298. doi: 10.1016/S0006-3495(97)78353-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Steinhoff H., Savitsky A., Wegener C., Pfeiffer M., Plato M., Möbius K. High-field EPR studies of the structure and conformational changes of site-directed spin labeled bacteriorhodopsin. Biochim Biophys Acta. 2000 Apr 21;1457(3):253–262. doi: 10.1016/s0005-2728(00)00106-7. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Subramaniam S., Henderson R. Molecular mechanism of vectorial proton translocation by bacteriorhodopsin. Nature. 2000 Aug 10;406(6796):653–657. doi: 10.1038/35020614. [DOI] [PubMed] [Google Scholar]
  32. Subramaniam S., Lindahl M., Bullough P., Faruqi A. R., Tittor J., Oesterhelt D., Brown L., Lanyi J., Henderson R. Protein conformational changes in the bacteriorhodopsin photocycle. J Mol Biol. 1999 Mar 19;287(1):145–161. doi: 10.1006/jmbi.1999.2589. [DOI] [PubMed] [Google Scholar]
  33. Thorgeirsson T. E., Xiao W., Brown L. S., Needleman R., Lanyi J. K., Shin Y. K. Transient channel-opening in bacteriorhodopsin: an EPR study. J Mol Biol. 1997 Nov 14;273(5):951–957. doi: 10.1006/jmbi.1997.1362. [DOI] [PubMed] [Google Scholar]
  34. Tiebel B., Radzwill N., Aung-Hilbrich L. M., Helbl V., Steinhoff H. J., Hillen W. Domain motions accompanying Tet repressor induction defined by changes of interspin distances at selectively labeled sites. J Mol Biol. 1999 Jul 2;290(1):229–240. doi: 10.1006/jmbi.1999.2875. [DOI] [PubMed] [Google Scholar]
  35. Vonck J. A three-dimensional difference map of the N intermediate in the bacteriorhodopsin photocycle: part of the F helix tilts in the M to N transition. Biochemistry. 1996 May 7;35(18):5870–5878. doi: 10.1021/bi952663c. [DOI] [PubMed] [Google Scholar]
  36. Vonck J. Structure of the bacteriorhodopsin mutant F219L N intermediate revealed by electron crystallography. EMBO J. 2000 May 15;19(10):2152–2160. doi: 10.1093/emboj/19.10.2152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Váró G., Lanyi J. K. Kinetic and spectroscopic evidence for an irreversible step between deprotonation and reprotonation of the Schiff base in the bacteriorhodopsin photocycle. Biochemistry. 1991 May 21;30(20):5008–5015. doi: 10.1021/bi00234a024. [DOI] [PubMed] [Google Scholar]
  38. Xiao W., Brown L. S., Needleman R., Lanyi J. K., Shin Y. K. Light-induced rotation of a transmembrane alpha-helix in bacteriorhodopsin. J Mol Biol. 2000 Dec 15;304(5):715–721. doi: 10.1006/jmbi.2000.4255. [DOI] [PubMed] [Google Scholar]
  39. Zimányi L., Váró G., Chang M., Ni B., Needleman R., Lanyi J. K. Pathways of proton release in the bacteriorhodopsin photocycle. Biochemistry. 1992 Sep 15;31(36):8535–8543. doi: 10.1021/bi00151a022. [DOI] [PubMed] [Google Scholar]

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