Characterization of the conformational change in the M1 and M2 substates of bacteriorhodopsin by the combined use of visible and infrared spectroscopy.

A combination of visible and Fourier transform infrared (FTIR) spectroscopies is used to characterize the formation of the M1 and M2 substates of the bacteriorhodopsin photocycle in glucose-embedded, hydrated thin films. Difference FTIR bands in the amide I region verify the previously reported existence of a significant peptide backbone conformational change in the transition from M1 to M2. The visible absorption spectra demonstrate that contamination of the M-intermediate samples by L, N, or other non-M species should contribute negligibly to the observed changes in the amide I region, and this conclusion is supported by comparison of specific carboxyl group peaks with corresponding bands in published L and N FTIR difference spectra. Based upon spectroscopic results, an extension of the C-T Model (Fodor, S., Ames, J., Gebhard, R., van den Berg, E., Stoeckenius, W., Lugtenberg, J., and Mathies, R. (1988) Biochemistry 27, 7097-7101) is presented. The results of this work suggest that protein structural changes should be clearly visible in M-bR, difference Fourier density maps and that these structural changes may in turn elucidate how bacteriorhodopsin actively pumps ions across the purple membrane of Halobacterium halobium.

[1]  S. O. Smith,et al.  Vibrational analysis of the all-trans retinal protonated Schiff base. , 1985, Biophysical journal.

[2]  J. Lanyi,et al.  Distortions in the photocycle of bacteriorhodopsin at moderate dehydration. , 1991, Biophysical journal.

[3]  D. Oesterhelt,et al.  Time‐resolved X‐ray diffraction study of structural changes associated with the photocycle of bacteriorhodopsin. , 1991, The EMBO journal.

[4]  S. P. Fodor,et al.  Bacteriorhodopsin's M412 intermediate contains a 13-cis, 14-s-trans, 15-anti-retinal Schiff base chromophore. , 1989, Biochemistry.

[5]  S. Chekalin,et al.  Primary events in bacteriorhodopsin probed by subpicosecond spectroscopy , 1985 .

[6]  Daniel E. Koshland,et al.  Protein Shape and Biological Control , 1973 .

[7]  R. Lozier,et al.  Flash spectroscopy of purple membrane. , 1987, Biophysical journal.

[8]  H. Khorana,et al.  Vibrational spectroscopy of bacteriorhodopsin mutants: light-driven proton transport involves protonation changes of aspartic acid residues 85, 96, and 212. , 1988, Biochemistry.

[9]  C. H. Brito Cruz,et al.  Direct observation of the femtosecond excited-state cis-trans isomerization in bacteriorhodopsin. , 1988, Science.

[10]  R. Mathies,et al.  Resonance Raman evidence for an all-trans to 13-cis isomerization in the proton-pumping cycle of bacteriorhodopsin. , 1980, Biochemistry.

[11]  T. Earnest,et al.  Orientation of the bacteriorhodopsin chromophore probed by polarized Fourier transform infrared difference spectroscopy. , 1986, Biochemistry.

[12]  J. Lanyi,et al.  THE TWO CONSECUTIVE M SUBSTATES IN THE PHOTOCYCLE OF BACTERIORHODOPSIN ARE AFFECTED SPECIFICALLY BY THE D85N AND D96N RESIDUE REPLACEMENTS , 1992, Photochemistry and photobiology.

[13]  S. P. Fodor,et al.  Chromophore structure in bacteriorhodopsin's N intermediate: implications for the proton-pumping mechanism. , 1988, Biochemistry.

[14]  T. Earnest,et al.  Polarized Fourier transform infrared spectroscopy of bacteriorhodopsin. Transmembrane alpha helices are resistant to hydrogen/deuterium exchange. , 1990, Biophysical journal.

[15]  B. Hess,et al.  Simultaneous monitoring of light-induced changes in protein side-group protonation, chromophore isomerization, and backbone motion of bacteriorhodopsin by time-resolved Fourier-transform infrared spectroscopy. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[16]  P. Ormos.  Infrared spectroscopic demonstration of a conformational change in bacteriorhodopsin involved in proton pumping. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[17]  J. Lanyi,et al.  Kinetic and spectroscopic evidence for an irreversible step between deprotonation and reprotonation of the Schiff base in the bacteriorhodopsin photocycle. , 1991, Biochemistry.

[18]  H. Khorana,et al.  Vibrational spectroscopy of bacteriorhodopsin mutants. Evidence that ASP-96 deprotonates during the M----N transition. , 1991, The Journal of biological chemistry.

[19]  J. Lanyi,et al.  Structures of aspartic acid-96 in the L and N intermediates of bacteriorhodopsin: analysis by Fourier transform infrared spectroscopy. , 1992, Biochemistry.

[20]  J. Nagle,et al.  PHOTOCYCLE KINETICS: ANALYSIS OF RAMAN DATA FROM BACTERIORHODOPSIN * , 1991, Photochemistry and photobiology.

[21]  J. Lanyi,et al.  Thermodynamics and energy coupling in the bacteriorhodopsin photocycle. , 1991, Biochemistry.

[22]  Y. Amemiya,et al.  Crystallographic characterization by X‐ray diffraction of the M‐intermediate from the photo‐cycle of bacteriorhodopsin at room temperature , 1991, FEBS letters.

[23]  W. Stoeckenius,et al.  Tunable laser resonance raman spectroscopy of bacteriorhodopsin. , 1974, Proceedings of the National Academy of Sciences of the United States of America.

[24]  R. Mathies,et al.  Chromophore structure in bacteriorhodopsin's O640 photointermediate , 1983 .

[25]  J. Lanyi,et al.  Water is required for proton transfer from aspartate-96 to the bacteriorhodopsin Schiff base. , 1991, Biochemistry.

[26]  Shuo-liang Lin,et al.  FTIR difference studies on apoproteins. Protonation states of aspartic and glutamic acid residues during the photocycle of bacteriorhodopsin , 1987 .

[27]  P. Ahl,et al.  Millisecond Fourier-transform infrared difference spectra of bacteriorhodopsin's M412 photoproduct. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[28]  T. Yoshizawa,et al.  Fourier transform infrared study of the N intermediate of bacteriorhodopsin. , 1991, Biochemistry.

[29]  B Hess,et al.  Light-driven protonation changes of internal aspartic acids of bacteriorhodopsin: an investigation by static and time-resolved infrared difference spectroscopy using [4-13C]aspartic acid labeled purple membrane. , 1985, Biochemistry.

[30]  R A Mathies,et al.  From femtoseconds to biology: mechanism of bacteriorhodopsin's light-driven proton pump. , 1991, Annual review of biophysics and biophysical chemistry.

[31]  J. Lanyi,et al.  A residue substitution near the beta-ionone ring of the retinal affects the M substates of bacteriorhodopsin. , 1992, Biophysical journal.

[32]  R. Mathies,et al.  Resonance Raman spectra of bacteriorhodopsin's primary photoproduct: evidence for a distorted 13-cis retinal chromophore. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[33]  K. Rothschild,et al.  Protein dynamics in the bacteriorhodopsin photocycle: submillisecond Fourier transform infrared spectra of the L, M, and N photointermediates. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[34]  B. Hess,et al.  Hydration effects on the photocycle of bacteriorhodopsin in thin layers of purple membrane , 1977, Nature.

[35]  J. Lanyi,et al.  Protonation and deprotonation of the M, N, and O intermediates during the bacteriorhodopsin photocycle. , 1990, Biochemistry.

[36]  D. Oesterhelt,et al.  Rhodopsin-like protein from the purple membrane of Halobacterium halobium. , 1971, Nature: New biology.

[37]  T G Ebrey,et al.  Ultraviolet and visible absorption spectra of the purple membrane protein and the photocycle intermediates. , 1978, Biochemistry.

[38]  P. Tavan,et al.  Structural investigation of bacteriorhodopsin and some of its photoproducts by polarized Fourier transform infrared spectroscopic methods-difference spectroscopy and photoselection. , 1991, Biophysical journal.

[39]  B. Hess,et al.  Role of aspartate-96 in proton translocation by bacteriorhodopsin. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[40]  R. Mathies,et al.  The role of back-reactions and proton uptake during the N----O transition in bacteriorhodopsin's photocycle: a kinetic resonance Raman study. , 1990, Biochemistry.

[41]  G. Zaccai,et al.  Structural changes in bacteriorhodopsin during proton translocation revealed by neutron diffraction. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[42]  R. Henderson,et al.  Electron diffraction analysis of the M412 intermediate of bacteriorhodopsin. , 1986, Biophysical journal.

[43]  P. Roepe,et al.  Tyrosine and carboxyl protonation changes in the bacteriorhodopsin photocycle. 1. M412 and L550 intermediates. , 1987, Biochemistry.