X-ray Structure of Bacteriorhodopsin at 2.5 Angstroms from Microcrystals Grown in Lipidic Cubic Phases

American Association for the Advancement of Science (AAAS) - Tập 277 Số 5332 - Trang 1676-1681 - 1997
Eva Pebay‐Peyroula1,2, Gabriele Rummel1,2, Jürg P. Rosenbusch1,2, Ehud M. Landau1,2
1E. Pebay-Peyroula, Institut de Biologie Structurale/CEA-CNRS/Université Joseph Fourier, 41 Avenue des Martyrs, F-38027 Grenoble Cedex 1, France.
2G. Rummel, J. P. Rosenbusch, E. M. Landau, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.

Tóm tắt

Lipidic cubic phases provide a continuous three-dimensional bilayer matrix that facilitates nucleation and growth of bacteriorhodopsin microcrystals. The crystals diffract x-rays isotropically to 2.0 angstroms. The structure of this light-driven proton pump was solved at a resolution of 2.5 angstroms by molecular replacement, using previous results from electron crystallographic studies as a model. The earlier structure was generally confirmed, but several differences were found, including loop conformations and side chain residues. Eight water molecules are now identified experimentally in the proton pathway. These findings reveal the constituents of the proton translocation pathway in the ground state.

Từ khóa


Tài liệu tham khảo

D. Oesterhelt J. Tittor E. Bamberg J. Bioenerg. Biomem. 24 181 (1992).

Oesterhelt D., Stoeckenius W., Proc. Natl. Acad. Sci. U.S.A. 70, 2853 (1973).

10.1038/257028a0

Unwin P. N. T., Henderson R., J. Mol. Biol. 94, 425 (1975).

Henderson R., J. Mol. Biol. 93, 123 (1975);

; A. E. Blaurock ibid. p. 139.

Henderson R., et al., ibid. 213, 899 (1990).

N. Grigorieff T. A. Ceska K. H. Downing J. M. Baldwin R. Henderson ibid. 259 393 (1996).

Engelman D. M., Henderson R., McLachlan A. D., Wallace B. A., Proc. Natl. Acad. Sci. U.S.A. 77, 2023 (1980).

Ovchinnikov Y. A., Abdulaev N. G., Feigina M. Y., Kiselev A. V., Lobanov N. A., FEBS Lett. 100, 219 (1979);

; H. G. Khorana et al. Proc. Natl. Acad. Sci. U.S.A. 76 5046 (1979);

Khorana H. G., J. Biol. Chem. 263, 7439 (1988);

Dencher N. A., Dresselhaus D., Zaccai G., Büldt G., Proc. Natl. Acad. Sci. U.S.A. 86, 7876 (1989);

Ni B. F., Chang M., Duschl A., Lanyi J., Needleman R., Gene 90, 169 (1990);

Krebs M. P., Hauss T., Heyn M. P., Rajbhandary U. L., Khorana H. G., Proc. Natl. Acad. Sci. U.S.A. 88, 859 (1991);

; R. A. Mathies S. W. Lin J. B. Ames W. T. Pollard Annu. Rev. Biophys. Chem. 20 491 (1991);

Sasaki J., Shichida Y., Lanyi J. K., Maeda A., J. Biol. Chem. 267, 20782 (1992);

Lanyi J. K., Biochim. Biophys. Acta 1183, 241 (1993);

Ferrand M., et al., FEBS Lett. 327, 256 (1993) ;

Xu D., Sheves M., Schulten K., Biophys. J. 69, 2745 (1995).

Gerwert K., Souvignier G., Hess B., Proc. Natl. Acad. Sci. U.S.A. 87, 9774 (1990).

Humphrey W., Logunov I., Schulten K., Sheves M., Biochemistry 33, 3668 (1994).

Henderson R., Shotton D., J. Mol. Biol. 139, 99 (1980);

Michel H., Oesterhelt D., Proc. Natl. Acad. Sci U.S.A. 77, 1283 (1980);

Michel H., EMBO J. 1, 1267 (1982);

Schertler G. F. X., Bartunik H. D., Michel H., Oesterhelt D., J. Mol. Biol. 234, 156 (1993).

10.1073/pnas.93.25.14532

. Monoolein (1-monooleoyl- rac -glycerol C 18:1c9 ) was obtained from Sigma.

E. M. Landau G. Rummel S. W. Cowan-Jacob J. P. Rosenbusch J. Phys. Chem. B 101 1935 (1997).

At room temperature a powder pattern with one strong diffraction ring at 53 Å (accessible low-resolution limit is 60 Å) was observed. Upon rapid cooling to liquid nitrogen temperature the diffraction image changed to multiple low-resolution rings at 52 Å 25 Å 12 Å and a strong ring at 4.5 Å. The latter is also observed at 4°C and can be assigned to the periodicity of the lipid chain packing [

Wilkins M. H. F., Blaurock A. E., Engelman D. M., Nature New Biol. 230, 72 (1971);

]. This transition also manifests itself by the fact that the cubic phase optically transparent at room temperature turns slightly opaque upon cooling.

The rms deviations are 0.7 and 1.2 Å for the main chain and for all the non-hydrogen atoms respectively if only the helices are considered. This compares with rms deviations of 3.6 Å (main chain) and 4.0 Å (all non-hydrogen atoms) if all residues between 7 and 225 are included.

The degree of distortion was unclear. A recent nuclear magnetic resonance study claims that the angles for the C18 C19 and C20 methyl groups with the membrane normal are 37° 40° and 32° respectively [

Ulrich A. S., Watts A., Wallat I., Heyn M. P., Biochemistry 33, 5370 (1994);

] whereas the EM structure represents them as 38.6° 29.3° and 16.1° respectively. Our structure reveals values of 32° 34° and 10° respectively.

Schulten K., Tavan P., Nature 272, 85 (1978).

Scharnagl C., Hettenkofer J., Fischer S. F., J. Phys. Chem. 99, 7787 (1995).

Brown L. S., et al., J. Biol. Chem. 270, 27122 (1995).

Papadopoulos G., Dencher N. A., Zaccai G., Büldt G., J. Mol. Biol. 214, 15 (1990).

M. S. Braiman et al. Biochemistry 27 8516 (1988).

Le Coutre J., Gerwert K., FEBS Lett. 398, 333 (1996).

Takei H., Gat Y., Rothman Z., Lewis A., Sheves M., J. Biol. Chem. 269, 7387 (1994).

Tittor J., Schweiger U., Oesterhelt D., Bamberg E., Biophys. J. 67, 1682 (1994).

Riesle J., Oesterhelt D., Dencher N. A., Heberle J., Biochemistry 35, 6635 (1996).

Szaraz S., Oesterhelt D., Ormos P., Biophys. J. 67, 1706 (1994).

J. Heberle and E. M. Landau unpublished results.

Lindblom G., Rilfors L., Biochim. Biophys. Acta 988, 221 (1989).

Weissbuch I., Popovitz-Biro R., Lahav M., Leiserowitz L., Acta Crystallogr. B51, 115 (1995);

10.1126/science.276.5312.575

Z. Otwinowski in Data Collection and Processing L. Sawyer N. Isaacs S. Bailey Eds. (SERC Daresbury Laboratory Warrington UK 1993) pp. 56–62.

Collaborative Computation Project 4 Acta Crystallogr. D50 760 (1994).

Jones A. T., Zou J. Y., Cowan S. W., Kjeldgaard M., ibid. A47, 110 (1991).

A. T. Brünger X-PLOR Version 3.1: A System for X-ray Crystallography and NMR (Yale Univ. Press New Haven CT 1992).

We thank C. Riekel H. Belrahli and A. Bram for invaluable help on the ID13 beamline; E. Fanchon R. Kahn and M. Roth on the French beamline D2AM; G. Büldt for his generosity; G. Büldt S. Cusack O. Dideberg J. Heberle J. Sass and G. Zaccai for stimulating discussions; and T. Bickle and R. Eisenberg for critically reading the manuscript. Supported by the Université Joseph Fourier the Institut de Biologie Structurale the Institut Universitaire de France the University of Basel and grants from the Swiss National Science Foundation (SPP 5002-37911 and -46092) EU-BIOMED (BIO4CT96/BBW95.0137.2) and EU-BIOTECH (EU-BIO4CT96/BBW96.0145.1). Beam time at the ESRF (Grenoble France) was granted under experiments LS-435 LS-553 LS-655 02.03.060 and 02.03.111.