Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 2002 May;82(5):2610–2616. doi: 10.1016/S0006-3495(02)75602-6

Time-resolved X-ray diffraction reveals movement of F helix of D96N bacteriorhodopsin during M-MN transition at neutral pH.

Toshihiko Oka 1, Naoto Yagi 1, Fumio Tokunaga 1, Mikio Kataoka 1
PMCID: PMC1302049  PMID: 11964247

Abstract

D96N bacteriorhodopsin has two photointermediates with the deprotonated Schiff base: the M and MN intermediates. We measure the time-resolved x-ray diffraction of the D96N purple membrane after flash photoexcitation (pH 7.0, 25 degrees C). The data clearly show the M-MN transition during the D96N photocycle. Low-resolution projection maps of these states show that the F helix of the MN intermediate shifts from its original position and this shift is much larger than that of the M intermediate. This indicates that the F helix moves in the M-MN transition of the D96N bacteriorhodopsin photocycle. Moreover, the existence of the MN intermediate in the D96N photocycle under neutral pH indicates that the MN intermediate is not peculiar to the alkaline condition. It is notable that the structural transition of M-MN is independent of the protonation state of the Schiff base. Therefore, the F helix movement precedes reprotonation of the Schiff base in the bacteriorhodopsin photocycle. Our previous study showed that the M-MN transition is hydration-dependent and that the MN intermediate is more hydrated than the M intermediate. Considering this together with the present results, we conclude that the movement of the F helix causes hydration of the cytoplasmic side, which promotes the reprotonation of the Schiff base.

Full Text

The Full Text of this article is available as a PDF (211.2 KB).

Selected References

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

  1. 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]
  2. Edman K., Nollert P., Royant A., Belrhali H., Pebay-Peyroula E., Hajdu J., Neutze R., Landau E. M. High-resolution X-ray structure of an early intermediate in the bacteriorhodopsin photocycle. Nature. 1999 Oct 21;401(6755):822–826. doi: 10.1038/44623. [DOI] [PubMed] [Google Scholar]
  3. Ferrand M., Dianoux A. J., Petry W., Zaccaï G. Thermal motions and function of bacteriorhodopsin in purple membranes: effects of temperature and hydration studied by neutron scattering. Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9668–9672. doi: 10.1073/pnas.90.20.9668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ganea C., Gergely C., Ludmann K., Váró G. The role of water in the extracellular half channel of bacteriorhodopsin. Biophys J. 1997 Nov;73(5):2718–2725. doi: 10.1016/S0006-3495(97)78300-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Henderson R., Baldwin J. M., Ceska T. A., Zemlin F., Beckmann E., Downing K. H. Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. J Mol Biol. 1990 Jun 20;213(4):899–929. doi: 10.1016/S0022-2836(05)80271-2. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Kamikubo H., Oka T., Imamoto Y., Tokunaga F., Lanyi J. K., Kataoka M. The last phase of the reprotonation switch in bacteriorhodopsin: the transition between the M-type and the N-type protein conformation depends on hydration. Biochemistry. 1997 Oct 7;36(40):12282–12287. doi: 10.1021/bi9712302. [DOI] [PubMed] [Google Scholar]
  8. Kataoka M., Kamikubo H., Tokunaga F., Brown L. S., Yamazaki Y., Maeda A., Sheves M., Needleman R., Lanyi J. K. Energy coupling in an ion pump. The reprotonation switch of bacteriorhodopsin. J Mol Biol. 1994 Nov 4;243(4):621–638. doi: 10.1016/0022-2836(94)90037-x. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Lozier R. H., Bogomolni R. A., Stoeckenius W. Bacteriorhodopsin: a light-driven proton pump in Halobacterium Halobium. Biophys J. 1975 Sep;15(9):955–962. doi: 10.1016/S0006-3495(75)85875-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Luecke H., Schobert B., Cartailler J. P., Richter H. T., Rosengarth A., Needleman R., Lanyi J. K. Coupling photoisomerization of retinal to directional transport in bacteriorhodopsin. J Mol Biol. 2000 Jul 28;300(5):1237–1255. doi: 10.1006/jmbi.2000.3884. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Luecke H., Schobert B., Richter H. T., Cartailler J. P., Lanyi J. K. Structure of bacteriorhodopsin at 1.55 A resolution. J Mol Biol. 1999 Aug 27;291(4):899–911. doi: 10.1006/jmbi.1999.3027. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Needleman R., Chang M., Ni B., Váró G., Fornés J., White S. H., Lanyi J. K. Properties of Asp212----Asn bacteriorhodopsin suggest that Asp212 and Asp85 both participate in a counterion and proton acceptor complex near the Schiff base. J Biol Chem. 1991 Jun 25;266(18):11478–11484. [PubMed] [Google Scholar]
  16. Ni B. F., Chang M., Duschl A., Lanyi J., Needleman R. An efficient system for the synthesis of bacteriorhodopsin in Halobacterium halobium. Gene. 1990 May 31;90(1):169–172. doi: 10.1016/0378-1119(90)90456-2. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Oka T., Yagi N., Fujisawa T., Kamikubo H., Tokunaga F., Kataoka M. Time-resolved x-ray diffraction reveals multiple conformations in the M-N transition of the bacteriorhodopsin photocycle. Proc Natl Acad Sci U S A. 2000 Dec 19;97(26):14278–14282. doi: 10.1073/pnas.260504897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Royant A., Edman K., Ursby T., Pebay-Peyroula E., Landau E. M., Neutze R. Helix deformation is coupled to vectorial proton transport in the photocycle of bacteriorhodopsin. Nature. 2000 Aug 10;406(6796):645–648. doi: 10.1038/35020599. [DOI] [PubMed] [Google Scholar]
  22. Rödig C., Siebert F. Distortion of the L-->M transition in the photocycle of the bacteriorhodopsin mutant D96N: a time-resolved step-scan FTIR investigation. FEBS Lett. 1999 Feb 19;445(1):14–18. doi: 10.1016/s0014-5793(99)00088-5. [DOI] [PubMed] [Google Scholar]
  23. Sasaki J., Shichida Y., Lanyi J. K., Maeda A. Protein changes associated with reprotonation of the Schiff base in the photocycle of Asp96-->Asn bacteriorhodopsin. The MN intermediate with unprotonated Schiff base but N-like protein structure. J Biol Chem. 1992 Oct 15;267(29):20782–20786. [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. 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]
  27. Tokaji Z. Cooperativity-regulated parallel pathways of the bacteriorhodopsin photocycle. FEBS Lett. 1995 Jan 3;357(2):156–160. doi: 10.1016/0014-5793(94)01344-z. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Váró G., Lanyi J. K. Distortions in the photocycle of bacteriorhodopsin at moderate dehydration. Biophys J. 1991 Feb;59(2):313–322. doi: 10.1016/S0006-3495(91)82225-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES