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. 1999 Apr;76(4):2183–2191. doi: 10.1016/S0006-3495(99)77373-X

Time-resolved absorption and photothermal measurements with sensory rhodopsin I from Halobacterium salinarum.

A Losi 1, S E Braslavsky 1, W Gärtner 1, J L Spudich 1
PMCID: PMC1300190  PMID: 10096912

Abstract

An expansion accompanying the formation of the first intermediate in the photocycle of transducer-free sensory rhodopsin I (SRI) was determined by means of time-resolved laser-induced optoacoustic spectroscopy. For the native protein (SRI-WT), the absolute value of the expansion is approximately 5.5 mL and for the mutant SRI-D76N, approximately 1.5 mL per mol of phototransformed species (in 0.5 M NaCl), calculated by using the formation quantum yield for the first intermediate (S610) of Phi610 = 0.4 +/- 0.05 for SRI-WT and 0.5 +/- 0.05 for SRI-D76N, measured by laser-induced optoacoustic spectroscopy and by laser flash photolysis. The similarity in Phi610 and in the determined value of the energy level of S610, E610 = (142 +/- 12) kJ/mol for SRI-WT and SRI-D76N indicates that Asp76 is not directly involved in the first step of the phototransformation. The increase with pH of the magnitude of the structural volume change for the formation of S610 in SRI-WT and in SRI-D76N upon excitation with 580 nm indicates also that amino acids other than Asp76, and other than those related to the Schiff base, are involved in the process. The difference in structural volume changes as well as differences in the activation parameters for the S610 decay should be attributed to differences in the rigidity of the cavity surrounding the chromophore. Except for the decay of the first intermediate, which is faster than in the SRI-transducer complex, the rate constants of the photocycle for transducer-free SRI in detergent suspension are strongly retarded with respect to wild-type membranes (this comparison should be done with great care because the preparation of both samples is very different).

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Selected References

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  1. Bogomolni R. A., Spudich J. L. The photochemical reactions of bacterial sensory rhodopsin-I. Flash photolysis study in the one microsecond to eight second time window. Biophys J. 1987 Dec;52(6):1071–1075. doi: 10.1016/S0006-3495(87)83301-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bogomolni R. A., Stoeckenius W., Szundi I., Perozo E., Olson K. D., Spudich J. L. Removal of transducer HtrI allows electrogenic proton translocation by sensory rhodopsin I. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):10188–10192. doi: 10.1073/pnas.91.21.10188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Butt H. J., Fendler K., Bamberg E., Tittor J., Oesterhelt D. Aspartic acids 96 and 85 play a central role in the function of bacteriorhodopsin as a proton pump. EMBO J. 1989 Jun;8(6):1657–1663. doi: 10.1002/j.1460-2075.1989.tb03556.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Drachev L. A., Kaulen A. D., Skulachev V. P. Time resolution of the intermediate steps in the bacteriorhodopsin-linked electrogenesis. FEBS Lett. 1978 Mar 1;87(1):161–167. doi: 10.1016/0014-5793(78)80157-4. [DOI] [PubMed] [Google Scholar]
  5. Dunitz J. D. Win some, lose some: enthalpy-entropy compensation in weak intermolecular interactions. Chem Biol. 1995 Nov;2(11):709–712. doi: 10.1016/1074-5521(95)90097-7. [DOI] [PubMed] [Google Scholar]
  6. Haupts U., Bamberg E., Oesterhelt D. Different modes of proton translocation by sensory rhodopsin I. EMBO J. 1996 Apr 15;15(8):1834–1841. [PMC free article] [PubMed] [Google Scholar]
  7. Haupts U., Eisfeld W., Stockburger M., Oesterhelt D. Sensory rhodopsin I photocycle intermediate SRI380 contains 13-cis retinal bound via an unprotonated Schiff base. FEBS Lett. 1994 Dec 12;356(1):25–29. doi: 10.1016/0014-5793(94)01226-1. [DOI] [PubMed] [Google Scholar]
  8. Hoff W. D., Jung K. H., Spudich J. L. Molecular mechanism of photosignaling by archaeal sensory rhodopsins. Annu Rev Biophys Biomol Struct. 1997;26:223–258. doi: 10.1146/annurev.biophys.26.1.223. [DOI] [PubMed] [Google Scholar]
  9. Krebs M. P., Spudich E. N., Spudich J. L. Rapid high-yield purification and liposome reconstitution of polyhistidine-tagged sensory rhodopsin I. Protein Expr Purif. 1995 Dec;6(6):780–788. doi: 10.1006/prep.1995.0009. [DOI] [PubMed] [Google Scholar]
  10. Lin S. L., Yan B. Three-dimensional model of sensory rhodopsin I reveals important restraints between the protein and the chromophore. Protein Eng. 1997 Mar;10(3):197–206. doi: 10.1093/protein/10.3.197. [DOI] [PubMed] [Google Scholar]
  11. Lindemann P., Braslavsky S. E., Cordonnier M. M., Pratt L. H., Schaffner K. Effects of bound monoclonal antibodies on the decay of the phototransformation intermediates I700(1,2) from native Avena phytochrome. Photochem Photobiol. 1993 Sep;58(3):417–424. doi: 10.1111/j.1751-1097.1993.tb09584.x. [DOI] [PubMed] [Google Scholar]
  12. Olson K. D., Spudich J. L. Removal of the transducer protein from sensory rhodopsin I exposes sites of proton release and uptake during the receptor photocycle. Biophys J. 1993 Dec;65(6):2578–2585. doi: 10.1016/S0006-3495(93)81295-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Rath P., Olson K. D., Spudich J. L., Rothschild K. J. The Schiff base counterion of bacteriorhodopsin is protonated in sensory rhodopsin I: spectroscopic and functional characterization of the mutated proteins D76N and D76A. Biochemistry. 1994 May 10;33(18):5600–5606. doi: 10.1021/bi00184a032. [DOI] [PubMed] [Google Scholar]
  14. Rath P., Spudich E., Neal D. D., Spudich J. L., Rothschild K. J. Asp76 is the Schiff base counterion and proton acceptor in the proton-translocating form of sensory rhodopsin I. Biochemistry. 1996 May 28;35(21):6690–6696. doi: 10.1021/bi9600355. [DOI] [PubMed] [Google Scholar]
  15. Ruddat A., Schmidt P., Gatz C., Braslavsky S. E., Gärtner W., Schaffner K. Recombinant type A and B phytochromes from potato. Transient absorption spectroscopy. Biochemistry. 1997 Jan 7;36(1):103–111. doi: 10.1021/bi962012w. [DOI] [PubMed] [Google Scholar]
  16. Schulenberg P. J., Rohr M., Gärtner W., Braslavsky S. E. Photoinduced volume changes associated with the early transformations of bacteriorhodopsin: a laser-induced optoacoustic spectroscopy study. Biophys J. 1994 Mar;66(3 Pt 1):838–843. doi: 10.1016/s0006-3495(94)80860-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Small J. R., Libertini L. J., Small E. W. Analysis of photoacoustic waveforms using the nonlinear least squares method. Biophys Chem. 1992 Jan;42(1):29–48. doi: 10.1016/0301-4622(92)80005-p. [DOI] [PubMed] [Google Scholar]
  18. Spudich E. N., Spudich J. L. The photochemical reactions of sensory rhodopsin I are altered by its transducer. J Biol Chem. 1993 Aug 5;268(22):16095–16097. [PubMed] [Google Scholar]
  19. Spudich J. L., Bogomolni R. A. Mechanism of colour discrimination by a bacterial sensory rhodopsin. Nature. 1984 Dec 6;312(5994):509–513. doi: 10.1038/312509a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Strassburger J. M., Gärtner W., Braslavsky S. E. Volume and enthalpy changes after photoexcitation of bovine rhodopsin: laser-induced optoacoustic studies. Biophys J. 1997 May;72(5):2294–2303. doi: 10.1016/S0006-3495(97)78874-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Yan B., Xie A., Nienhaus G. U., Katsuta Y., Spudich J. L. Steric constraints in the retinal binding pocket of sensory rhodopsin I. Biochemistry. 1993 Sep 28;32(38):10224–10232. doi: 10.1021/bi00089a044. [DOI] [PubMed] [Google Scholar]
  22. Yao V. J., Spudich J. L. Primary structure of an archaebacterial transducer, a methyl-accepting protein associated with sensory rhodopsin I. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11915–11919. doi: 10.1073/pnas.89.24.11915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Zhang D., Mauzerall D. Volume and enthalpy changes in the early steps of bacteriorhodopsin photocycle studied by time-resolved photoacoustics. Biophys J. 1996 Jul;71(1):381–388. doi: 10.1016/S0006-3495(96)79235-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. van Brederode M. E., Gensch T., Hoff W. D., Hellingwerf K. J., Braslavsky S. E. Photoinduced volume change and energy storage associated with the early transformations of the photoactive yellow protein from Ectothiorhodospira halophila. Biophys J. 1995 Mar;68(3):1101–1109. doi: 10.1016/S0006-3495(95)80284-5. [DOI] [PMC free article] [PubMed] [Google Scholar]

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