Aug 26, 1985 - K. J. ROTHSCHILD*, P. ROEPE*, P.L. AHL*, T. N. EARNEST*, R. A. BOGOMOLNIt, S. K. DAS GUPTAt, .... payment. This article must therefore ...
Proc. Natl. Acad. Sci. USA Vol. 83, pp. 347-351, January 1986 Biophysics
Evidence for a tyrosine protonation change during the primary phototransition of bacteriorhodopsin at low temperature (purple membrane/Fourier transform infrared/ultraviolet/tyrosinate/proton transport)
K. J. ROTHSCHILD*, P. ROEPE*, P.L. AHL*, T. N. EARNEST*, R. A. BOGOMOLNIt, S. K. DAS GUPTAt, C. M. MULLIKEN§, AND J. HERZFELD§ *Departments of Physics and Physiology, Boston University, Boston, MA 02215; tDepartment of Biochemistry and Biophysics, University of California, San Francisco, CA 94113; §The Biophysical Laboratory, Department of Physiology, Harvard Medical School, Boston, MA 02115; and tThe Francis Bitter National Magnet Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02134
Communicated by M. A. El-Sayed, August 26, 1985
ABSTRACT Isotopically labeled tyrosines have been selectively incorporated into bacteriorhodopsin (bR). A comparison of the low-temperature bR570 to K Fourier transform infrared-difference spectra of these samples and normal bR provides information about the role of tyrosine in the primary phototransition. Several tyrosine contributions to the difference spectrum are found. These results and comparison with the spectra of model compounds suggest that a tyrosinate group protonates during the bR570 to K transition. This conclusion is strongly supported by the results of UV difference spectros-
MATERIALS AND METHODS L-[Ring-2H4]tyrosine and [1-13C]phenol were prepared according to procedures to be described elsewhere (D. M. Rice, B. A. Lewis, R. G. Griffin, S.K.D., and J.H., unpublished results). [1-13C]Phenol was converted to L-[ring-4-13C]tyrOsine by Erwinia herbicola cells cultured for high tyrosine phenol ligase activity (14) and was purified by recrystallization. All isotope substitutions were verified by NMR spectroscopy. Halobacterium halobium R1 was grown in a synthetic medium like that of Gochnauer and Kushner (15) except that the D-amino acids and the NH4Cl were omitted, and that L-[ring-2H4]tyrosine with trace L-[ring-2,3,5,6-3H]tyrosine (New England Nuclear) or L-[ring-4-13C]tyrosine with trace L-[ring-2,6-3H]tyrosine (New England Nuclear) was substituted for the corresponding unlabeled amino acid. Purple membrane was isolated by the method of Oesterhelt and Stoeckenius (16). Specific activity measurements indicated that 50-80% of the tyrosine residues were labeled in various preparations. Amino acid analysis showed that
