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. 2002 Apr;82(4):2156–2164. doi: 10.1016/S0006-3495(02)75562-8

Probing the proton channel and the retinal binding site of Natronobacterium pharaonis sensory rhodopsin II.

Johann P Klare 1, Georg Schmies 1, Igor Chizhov 1, Kazumi Shimono 1, Naoki Kamo 1, Martin Engelhard 1
PMCID: PMC1302009  PMID: 11916871

Abstract

The sensory rhodopsin II from Natronobacterium pharaonis (NpSRII) was mutated to try to create functional properties characteristic of bacteriorhodopsin (BR), the proton pump from Halobacterium salinarum. Key residues from the cytoplasmic and extracellular proton transfer channel of BR as well as from the retinal binding site were chosen. The single site mutants L40T, F86D, P183E, and T204A did not display altered function as determined by the kinetics of their photocycles. However, the photocycle of each of the subsequent multisite mutations L40T/F86D, L40T/F86D/P183E, and L40T/F86D/P183E/T204A was quite different from that of the wild-type protein. The reprotonation of the Schiff base could be accelerated approximately 300- to 400-fold, to approximately two to three times faster than the corresponding reaction in BR. The greatest effect is observed for the quadruple mutant in which Thr-204 is replaced by Ala. This result indicates that mutations affecting conformational changes of the protein might be of decisive importance for the creation of BR-like functional properties.

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Selected References

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

  1. Balashov S. P., Imasheva E. S., Ebrey T. G., Chen N., Menick D. R., Crouch R. K. Glutamate-194 to cysteine mutation inhibits fast light-induced proton release in bacteriorhodopsin. Biochemistry. 1997 Jul 22;36(29):8671–8676. doi: 10.1021/bi970744y. [DOI] [PubMed] [Google Scholar]
  2. Balashov S. P., Lu M., Imasheva E. S., Govindjee R., Ebrey T. G., Othersen B., 3rd, Chen Y., Crouch R. K., Menick D. R. The proton release group of bacteriorhodopsin controls the rate of the final step of its photocycle at low pH. Biochemistry. 1999 Feb 16;38(7):2026–2039. doi: 10.1021/bi981926a. [DOI] [PubMed] [Google Scholar]
  3. Balashov S. P. Protonation reactions and their coupling in bacteriorhodopsin. Biochim Biophys Acta. 2000 Aug 30;1460(1):75–94. doi: 10.1016/s0005-2728(00)00131-6. [DOI] [PubMed] [Google Scholar]
  4. Chizhov I., Chernavskii D. S., Engelhard M., Mueller K. H., Zubov B. V., Hess B. Spectrally silent transitions in the bacteriorhodopsin photocycle. Biophys J. 1996 Nov;71(5):2329–2345. doi: 10.1016/S0006-3495(96)79475-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chizhov I., Engelhard M. Temperature and halide dependence of the photocycle of halorhodopsin from Natronobacterium pharaonis. Biophys J. 2001 Sep;81(3):1600–1612. doi: 10.1016/S0006-3495(01)75814-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chizhov I., Schmies G., Seidel R., Sydor J. R., Lüttenberg B., Engelhard M. The photophobic receptor from Natronobacterium pharaonis: temperature and pH dependencies of the photocycle of sensory rhodopsin II. Biophys J. 1998 Aug;75(2):999–1009. doi: 10.1016/S0006-3495(98)77588-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dower W. J., Miller J. F., Ragsdale C. W. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. doi: 10.1093/nar/16.13.6127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Engelhard M., Scharf B., Siebert F. Protonation changes during the photocycle of sensory rhodopsin II from Natronobacterium pharaonis. FEBS Lett. 1996 Oct 21;395(2-3):195–198. doi: 10.1016/0014-5793(96)01041-1. [DOI] [PubMed] [Google Scholar]
  9. Higuchi R., Krummel B., Saiki R. K. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 1988 Aug 11;16(15):7351–7367. doi: 10.1093/nar/16.15.7351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  11. Hohenfeld I. P., Wegener A. A., Engelhard M. Purification of histidine tagged bacteriorhodopsin, pharaonis halorhodopsin and pharaonis sensory rhodopsin II functionally expressed in Escherichia coli. FEBS Lett. 1999 Jan 15;442(2-3):198–202. doi: 10.1016/s0014-5793(98)01659-7. [DOI] [PubMed] [Google Scholar]
  12. Iwamoto M., Shimono K., Sumi M., Kamo N. Positioning proton-donating residues to the Schiff-base accelerates the M-decay of pharaonis phoborhodopsin expressed in Escherichia coli. Biophys Chem. 1999 Jun 28;79(3):187–192. doi: 10.1016/s0301-4622(99)00054-x. [DOI] [PubMed] [Google Scholar]
  13. Klostermeier D., Seidel R., Reinstein J. Functional properties of the molecular chaperone DnaK from Thermus thermophilus. J Mol Biol. 1998 Jun 19;279(4):841–853. doi: 10.1006/jmbi.1998.1816. [DOI] [PubMed] [Google Scholar]
  14. Kolbe M., Besir H., Essen L. O., Oesterhelt D. Structure of the light-driven chloride pump halorhodopsin at 1.8 A resolution. Science. 2000 May 26;288(5470):1390–1396. doi: 10.1126/science.288.5470.1390. [DOI] [PubMed] [Google Scholar]
  15. Lanyi J. K., Luecke H. Bacteriorhodopsin. Curr Opin Struct Biol. 2001 Aug;11(4):415–419. doi: 10.1016/s0959-440x(00)00226-8. [DOI] [PubMed] [Google Scholar]
  16. Lu M., Balashov S. P., Ebrey T. G., Chen N., Chen Y., Menick D. R., Crouch R. K. Evidence for the rate of the final step in the bacteriorhodopsin photocycle being controlled by the proton release group: R134H mutant. Biochemistry. 2000 Mar 7;39(9):2325–2331. doi: 10.1021/bi992554o. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Luecke H., Schobert B., Lanyi J. K., Spudich E. N., Spudich J. L. Crystal structure of sensory rhodopsin II at 2.4 angstroms: insights into color tuning and transducer interaction. Science. 2001 Jul 12;293(5534):1499–1503. doi: 10.1126/science.1062977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Michel H., Oesterhelt D. Electrochemical proton gradient across the cell membrane of Halobacterium halobium: effect of N,N'-dicyclohexylcarbodiimide, relation to intracellular adenosine triphosphate, adenosine diphosphate, and phosphate concentration, and influence of the potassium gradient. Biochemistry. 1980 Sep 30;19(20):4607–4614. doi: 10.1021/bi00561a011. [DOI] [PubMed] [Google Scholar]
  22. Radzwill N., Gerwert K., Steinhoff H. J. Time-resolved detection of transient movement of helices F and G in doubly spin-labeled bacteriorhodopsin. Biophys J. 2001 Jun;80(6):2856–2866. doi: 10.1016/S0006-3495(01)76252-2. [DOI] [PMC free article] [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. Royant A., Nollert P., Edman K., Neutze R., Landau E. M., Pebay-Peyroula E., Navarro J. X-ray structure of sensory rhodopsin II at 2.1-A resolution. Proc Natl Acad Sci U S A. 2001 Aug 14;98(18):10131–10136. doi: 10.1073/pnas.181203898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sasaki J., Spudich J. L. Proton circulation during the photocycle of sensory rhodopsin II. Biophys J. 1999 Oct;77(4):2145–2152. doi: 10.1016/S0006-3495(99)77055-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sasaki J., Spudich J. L. Proton transport by sensory rhodopsins and its modulation by transducer-binding. Biochim Biophys Acta. 2000 Aug 30;1460(1):230–239. doi: 10.1016/s0005-2728(00)00142-0. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Scharf B., Pevec B., Hess B., Engelhard M. Biochemical and photochemical properties of the photophobic receptors from Halobacterium halobium and Natronobacterium pharaonis. Eur J Biochem. 1992 Jun 1;206(2):359–366. doi: 10.1111/j.1432-1033.1992.tb16935.x. [DOI] [PubMed] [Google Scholar]
  29. Schmies G., Engelhard M., Wood P. G., Nagel G., Bamberg E. Electrophysiological characterization of specific interactions between bacterial sensory rhodopsins and their transducers. Proc Natl Acad Sci U S A. 2001 Jan 30;98(4):1555–1559. doi: 10.1073/pnas.031562298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schmies G., Lüttenberg B., Chizhov I., Engelhard M., Becker A., Bamberg E. Sensory rhodopsin II from the haloalkaliphilic natronobacterium pharaonis: light-activated proton transfer reactions. Biophys J. 2000 Feb;78(2):967–976. doi: 10.1016/S0006-3495(00)76654-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schäfer G., Engelhard M., Müller V. Bioenergetics of the Archaea. Microbiol Mol Biol Rev. 1999 Sep;63(3):570–620. doi: 10.1128/mmbr.63.3.570-620.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Seidel R., Scharf B., Gautel M., Kleine K., Oesterhelt D., Engelhard M. The primary structure of sensory rhodopsin II: a member of an additional retinal protein subgroup is coexpressed with its transducer, the halobacterial transducer of rhodopsin II. Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):3036–3040. doi: 10.1073/pnas.92.7.3036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Shimono K., Ikeura Y., Sudo Y., Iwamoto M., Kamo N. Environment around the chromophore in pharaonis phoborhodopsin: mutation analysis of the retinal binding site. Biochim Biophys Acta. 2001 Dec 1;1515(2):92–100. doi: 10.1016/s0005-2736(01)00394-7. [DOI] [PubMed] [Google Scholar]
  34. Shimono K., Iwamoto M., Sumi M., Kamo N. Effects of three characteristic amino acid residues of pharaonis phoborhodopsin on the absorption maximum. Photochem Photobiol. 2000 Jul;72(1):141–145. doi: 10.1562/0031-8655(2000)072<0141:eotcaa>2.0.co;2. [DOI] [PubMed] [Google Scholar]
  35. Shimono K., Iwamoto M., Sumi M., Kamo N. Functional expression of pharaonis phoborhodopsin in Escherichia coli. FEBS Lett. 1997 Dec 22;420(1):54–56. doi: 10.1016/s0014-5793(97)01487-7. [DOI] [PubMed] [Google Scholar]
  36. Spudich J. L. Variations on a molecular switch: transport and sensory signalling by archaeal rhodopsins. Mol Microbiol. 1998 Jun;28(6):1051–1058. doi: 10.1046/j.1365-2958.1998.00859.x. [DOI] [PubMed] [Google Scholar]
  37. Spudich J. L., Yang C. S., Jung K. H., Spudich E. N. Retinylidene proteins: structures and functions from archaea to humans. Annu Rev Cell Dev Biol. 2000;16:365–392. doi: 10.1146/annurev.cellbio.16.1.365. [DOI] [PubMed] [Google Scholar]
  38. Subramaniam S., Henderson R. Crystallographic analysis of protein conformational changes in the bacteriorhodopsin photocycle. Biochim Biophys Acta. 2000 Aug 30;1460(1):157–165. doi: 10.1016/s0005-2728(00)00136-5. [DOI] [PubMed] [Google Scholar]
  39. Sudo Y., Iwamoto M., Shimono K., Sumi M., Kamo N. Photo-induced proton transport of pharaonis phoborhodopsin (sensory rhodopsin II) is ceased by association with the transducer. Biophys J. 2001 Feb;80(2):916–922. doi: 10.1016/S0006-3495(01)76070-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Takao K., Kikukawa T., Araiso T., Kamo N. Azide accelerates the decay of M-intermediate of pharaonis phoborhodopsin. Biophys Chem. 1998 Jul 13;73(1-2):145–153. doi: 10.1016/s0301-4622(98)00156-2. [DOI] [PubMed] [Google Scholar]
  41. Wegener A. A., Chizhov I., Engelhard M., Steinhoff H. J. Time-resolved detection of transient movement of helix F in spin-labelled pharaonis sensory rhodopsin II. J Mol Biol. 2000 Aug 25;301(4):881–891. doi: 10.1006/jmbi.2000.4008. [DOI] [PubMed] [Google Scholar]
  42. Zhang W., Brooun A., McCandless J., Banda P., Alam M. Signal transduction in the archaeon Halobacterium salinarium is processed through three subfamilies of 13 soluble and membrane-bound transducer proteins. Proc Natl Acad Sci U S A. 1996 May 14;93(10):4649–4654. doi: 10.1073/pnas.93.10.4649. [DOI] [PMC free article] [PubMed] [Google Scholar]

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