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Site-directed isotope labelling and FTIR spectroscopy of bacteriorhodopsin

Nature Structural Biology volume 1, pages 512–517 (1994) Cite this article

Abstract

Insight into integral membrane proteins function is presently limited by the difficulty of producing three-dimensional crystals. In addition, X-ray structures of proteins normally do not provide information about the protonation state and structural changes of individual residues. We report here the first use of site-directed isotope labelling and Fourier transform infrared (FTIR) difference spectroscopy to detect structural changes at the level of single residues in an integral membrane protein. Two site-directed isotope labeled (SDIL) tyrosine analogues of bacteriorhodopsin were produced which exhibit normal activity. FTIR spectroscopy shows that out of 11 tyrosines, only Tyr 185 is structurally active during the early photocycle and may be part of a proton wire.

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References

  1. Stoeckenius, W. & Bogomolni, R.A. Bacteriorhodopsin and related pigments of halobacteria. A. Rev. Biochem. 51, 587–616 (1982)

    Article  CAS  Google Scholar 

  2. Khorana, H.G. Rhodopsin photoreceptor of the rod cell. An emerging pattern for structure and function. J. biol. Chem. 267, 1–4 (1992)

    CAS  PubMed  Google Scholar 

  3. Lanyi, J. Halorhodopsin, a light-driven electrogenic chloride-transport system. Physiol. Rev. 70, 319–329 (1990)

    Article  CAS  PubMed  Google Scholar 

  4. Spudich, J.L. & Bogomolni, R.A. Sensory Rhodopsins of Halobacteria. A. Rev. Biophys. biophys. Chem. 17, 193–215 (1988)

    Article  CAS  Google Scholar 

  5. Henderson, R. et al. Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. J. molec. Biol. 213, 899–929 (1990)

    Article  CAS  PubMed  Google Scholar 

  6. Shen, Y., Safinya, C.R., Liang, K.S., Ruppert, A.F. & Rothschild, K.J. Stabilization of the membrane protein bacteriorhodopsin to 140 °C in two dimensional films. Nature 366, 48–50 (1993)

    Article  CAS  Google Scholar 

  7. Lozier, R.H., Xie, A., Hofrichter, J. & Clore, G.M. Reversible steps in the bacteriorhodopsin photocycle. Proc. natn. Acad. Sci. U.S.A. 89, 3610–3614 (1992)

    Article  CAS  Google Scholar 

  8. Zundel, G. & Merz, H. Proton conduction in bacteriorhodopsin via a hydrogen-bonded chain with large proton polarizability. Biochem. biophys. Res. Commun. 101, 540 (1981)

    Article  PubMed  Google Scholar 

  9. Olejnik, J., Brzezinski, B. & Zundel, G. A proton pathway with large proton polarizability and the proton pumping mechanism in bacteriorhodopsin-fourier transform difference spectra of photoproducts of bacteriorhodpsin and of its pentademethyl analogue. J. molec. Struct. 271, 157–173 (1992)

    Article  CAS  Google Scholar 

  10. Nagle, J.F. & Mille, M. Molecular models of proton pumps. J. Chem. Phys. 74, 1367–1372 (1981)

    Article  CAS  Google Scholar 

  11. Rothschild, K.J., He, Y.W., Sonar, S., Marti, T. & Khorana, H.G. Vibrational spectroscopy of bacteriorhodopsin mutants. Evidence that Thr-46 and Thr-89 form part of a transient network of hydrogen bonds. J. biol. Chem. 267, 1615–1622 (1992)

    CAS  PubMed  Google Scholar 

  12. Rothschild, K.J. FTIR difference spectroscopy of bacteriorhodopsin: Toward a molecular model. J. Bioenerg. Biomembr. 24, 147–167 (1992)

    Article  CAS  PubMed  Google Scholar 

  13. Gerwert, K., Hess, B., Soppa, J. & Oesterhelt, D. Role of aspartate-96 in proton translocation by bacteriorhodopsin. Proc. natn. Acad. Sci. U.S.A. 86, 4943–4947 (1989)

    Article  CAS  Google Scholar 

  14. Bousché, O., Braiman, M., He, Y.W., Marti, T., Khorana, H.G. & Rothschild, K.J. Vibrational spectroscopy of bacteriorhodopsin mutants. Evidence that Asp-96 deprotonates during the M→N transition. J. biol. Chem. 266, 11063–11067 (1991)

    PubMed  Google Scholar 

  15. Rothschild, K.J. et al. Evidence for a tyrosine protonation change during the primary phototransition of bacteriorhodopsin at low temperature. Proc natn. Acad. Sci. U.S.A. 83, 347–351 (1986)

    Article  CAS  Google Scholar 

  16. Dollinger, G., Eisenstein, L., Lin, S.L., Nakanishi, K. & Termini, J. Fourier transform infrared difference spectroscopy of bacteriorhodopsin and its photoproducts regenerated with deuterated tyrosine. Biochemistry 25, 6524–6533 (1986)

    Article  CAS  PubMed  Google Scholar 

  17. Braiman, M.S. et al. Vibrational spectroscopy of bacteriorhodopsin mutants. I. Tyrosine-185 protonates and deprotonates during the photocycle. Proteins: Struct. Funct. Genet. 3, 219–229 (1988)

    Article  CAS  Google Scholar 

  18. He, Y.-W., Krebs, M.P., Herzfeld, J., Khorana, H.G. & Rothschild, K.J. FTIR spectroscopy, site directed mutagenesis and isotope labelling: A new approach for studying membrane proteins. Proc. SPIE-lnt. Soc. opt. Engng. 1575, 109–116 (1991)

    Google Scholar 

  19. Merz, H. & Zundel, G. Proton-transfer equilibriums in phenol-carboxylate hydrogen bonds. Implications for the mechanism of light-induced proton activation in bacteriorhodopsin. Chem. Phys. Lett. 95, 529–532 (1983)

    Article  CAS  Google Scholar 

  20. Braiman, M.S. et al. Vibrational spectroscopy of bacteriorhodopsin mutants: light-driven proton transport involves protonation changes of aspartic acid residues 85, 96, and 212. Biochemistry 27, 8516–8520 (1988)

    Article  CAS  PubMed  Google Scholar 

  21. Krebs, M.P. & Khorana, H.G. Mechanism of light-dependent proton translocation by bacteriorhodopsin. J. Bact. 175, 1555–1558 (1993)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Sonar, S., Krebs, M.P., Khorana, H.G. & Rothschild, K.J. Static and time-resolved absorption spectroscopy of the bacteriorhodopsin mutant Tyr-185→Phe: evidence for an equilibrium between bR570 and an O-like Species. Biochemistry 32, 2263–2271 (1993)

    Article  CAS  PubMed  Google Scholar 

  23. He, Y.-W., Krebs, M.P., Fischer, W.B., Khorana, H.G. & Rothschild, K.J. FTIR difference spectroscopy of the bacteriorhodopsin mutant Tyr-185→Phe: detection of a stable O-like species and characterization of its photocycle at low temperature. Biochemistry 32, 2282–2290 (1993)

    Article  CAS  PubMed  Google Scholar 

  24. Otto, H. et al. Substitution of amino acids Asp-85, Asp-212, and Arg-82 in bacteriorhodopsin affects the proton release phase of the pump and the pK of the Schiff base. Proc. natn. Acad. Sci. U.S.A. 87, 1018–1022 (1990)

    Article  CAS  Google Scholar 

  25. Subramaniam, S., Marti, T. & Khorana, H.G. Protonation state of Asp (Glu)-85 regulates the purple-to-blue transition in bacteriorhodopsin mutants Arg-82→Ala and Asp-85→Glu: the blue form is inactive in proton translocation. Proc. natn. Acad. Sci. U.S.A. 87, 1013–1017 (1990)

    Article  CAS  Google Scholar 

  26. Needleman, R. et al. Properties of Asp-212→Asn bacteriorhodopsin suggest that Asp-212 and Asp-85 both participate in a counterion and proton acceptor complex near the Schiff base. J. biol. Chem. 266, 11478–11484 (1991)

    CAS  PubMed  Google Scholar 

  27. Noren, C.J., Anthony-Cahill, S.J., Griffith, M.C. & Schultz, P.G. A general method for site-specific incorporation of unnatural amino acids into proteins. Science 244, 182–188 (1989)

    Article  CAS  PubMed  Google Scholar 

  28. Ellman, J.A., Mendel, D. & Schultz, P.G. Site-specific incorporation of novel backbone structures into proteins. Science 255, 197–200 (1992)

    Article  CAS  PubMed  Google Scholar 

  29. Bain, J., Glabe, C.G., Dix, T.A. & Chamberlin, R. Synthetic site-specific incorporation of a natural amino acid into a polypeptide. J. am. Chem. Soc. 111, 8013–8014 (1989)

    Article  CAS  Google Scholar 

  30. Bain, J.D. et al. Site-specific incorporation of nonnatural residues during in vitro protein biosynthesis with semisynthetic aminoacyl-tRNAs. Biochemistry 30, 5411–5421 (1991)

    Article  CAS  PubMed  Google Scholar 

  31. Sonar, S.M., Patel, N.P., Fischer, W. & Rothschild, K.J. Cell-free synthesis, functional refolding and spectroscopic characterization of an integral membrane protein: bacteriorhodopsin. Biochemistry 32, 13777–13781 (1993)

    Article  CAS  PubMed  Google Scholar 

  32. Kleina, J., Masson, J., Normally, J., Abelson, J. & Miller, J. Construction of Escherichia coli amber suppressor tRNA genes II. Synthesis of additional tRNA genes and improvement of suppressor efficiency. J. molec. Biol. 213, 705–717 (1990)

    Article  CAS  PubMed  Google Scholar 

  33. Normanly, J., Keleina, L., Masson, J., Abelson, J. & Miller, J. Construction of Escherichia coli amber suppressor tRNA genes III. Determination of tRNA Specificity. J. molec. Biol. 213, 719–726 (1990)

    Article  CAS  PubMed  Google Scholar 

  34. Seong, B.L. & RajBhandary, U.L. Mutants of Escherichia coli formylmethionine tRNA; A single base change enables initiator tRNA to act as an elongator in vitro. Proc. natn. Acad. Sci. U.S.A. 84, 8859–8863 (1987)

    Article  CAS  Google Scholar 

  35. Dunach, M. et al.Ultraviolet-visible transient spectroscopy of bacteriorhodopsin mutants. Evidence for two forms of tyrosine-185→phenylalanine. J. biol. Chem. 265, 16978–16984 (1990)

    CAS  PubMed  Google Scholar 

  36. Creuzet, F. et al. Determination of membrane protein structure by rotational resonance NMR: Bacteriorhodopsin. Science 251, 783–786 (1991)

    Article  CAS  PubMed  Google Scholar 

  37. Peersen, O.B., Yoshimura, S., Hojo, H., Aimoto, S. & Smith, S.O. Rotational resonance NMR measurements of intranuclear distances in an α-helical peptide. J. am. Chem. Soc. 114, 4332–4335 (1992)

    Article  CAS  Google Scholar 

  38. Lee, C.P., Seong, B.L. & RajBhandary, U.L. Structural and sequence elements important for recognition of E. coli formylmethionine tRNA by methionyl-tRNA transformylase are clustered in the acceptor stem. J. biol. Chem. 266, 18012–18018 (1991)

    CAS  PubMed  Google Scholar 

  39. RajBhandary, U.L. & Ghosh, H.P. Studies on polynucleotides XCI Yeast methionine transfer ribonucleic acid: purification, properties and terminal nucleotide sequences. J. biol. Chem. 244, 1104–1113 (1969)

    CAS  PubMed  Google Scholar 

  40. Rothschild, K.J. & Sonar, S. in CRC Handbook of Organic Photochemistry and Photobiology. (ed. Song, P.-S.) (CRC Press, London, 1994) (in the press).

    Google Scholar 

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Authors and Affiliations

  1. Physics Department and Molecular Biophysics Laboratory, Boston University, Boston, Massachusetts, 02215

    Sanjay Sonar, Matthew Coleman, Nilam Patel, Xiaomei Liu & Kenneth J. Rothschild

  2. Department of Biology Massachusetts, Institute of Technology, Cambridge, Massachusetts, 02139, USA

    Chan-Ping Lee & Uttam L. RajBhandary

  3. Department of Chemistry and Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, 02139, USA

    Thomas Marti & H. Gobind Khorana

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  1. Sanjay Sonar
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  2. Chan-Ping Lee
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Sonar, S., Lee, CP., Coleman, M. et al. Site-directed isotope labelling and FTIR spectroscopy of bacteriorhodopsin. Nat Struct Mol Biol 1, 512–517 (1994). https://doi.org/10.1038/nsb0894-512

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