MECHANISM OF PROTON PUMPING IN BACTERIORHODOPSIN BY SOLID-STATE NMR - THE PROTONATION STATE OF TYROSINE IN THE LIGHT-ADAPTED AND M-STATES

被引:36
作者
MCDERMOTT, AE
THOMPSON, LK
WINKEL, C
FARRAR, MR
PELLETIER, S
LUGTENBURG, J
HERZFELD, J
GRIFFIN, RG
机构
[1] MIT, FRANCIS BITTER NATL MAGNET LAB, CAMBRIDGE, MA 02139 USA
[2] MIT, DEPT CHEM, CAMBRIDGE, MA 02139 USA
[3] BRANDEIS UNIV, DEPT CHEM, WALTHAM, MA 02254 USA
[4] LEIDEN UNIV, DEPT CHEM, 2300 RA LEIDEN, NETHERLANDS
关键词
D O I
10.1021/bi00098a013
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Solid-state C-13 NMR spectra were employed to characterize the protonation state of tyrosine in the light-adapted (bR568) and M states of bacteriorhodopsin (bR). Difference spectra (isotopically labeled bR minus natural-abundance bR) were obtained for [4'-C-13]Tyr-labeled bR, regenerated with [14-C-13]retinal as an internal marker to identify the photocycle states. The [14-C-13]retinal has distinct chemical shifts for bR555, for bR568, and for the M intermediate generated and thermally trapped at pH 10 in the presence of 0.3 M KCl or 0.5 M guanidine. Previous work has demonstrated that tyrosine and tyrosinate are easily distinguished on the basis of the chemical shift of the 4'-C-13 label and that both NMR signals are detectable in dark-adapted bR, although the tyrosinate signal is only present at pH values greater than 12. In the present work, we show that neither the light-adapted form of bR prepared at pH 7 or 10 nor the M state thermally trapped at -80-degrees-C in 0.3 M KCl pH 10, or in 0.5 M guanidine pH 10, shows any detectable tyrosinate. In addition, after the M samples were briefly warmed (approximately 30 s), no tyrosinate was observed. However, small (1-2 ppm) changes in the structure or dispersion in the Tyr peak were observed in the M state phototrapped by either method. These changes were reversible when the sample was warmed, although on a time scale slower than the relaxation of the retinal back to the bR568 conformer. Such small changes may be interpreted as alterations in the environment or the hydrogen bonding of some of the tyrosines, but not as evidence for the formation of tyrosinate. In summary, these data indicate that no stable tyrosinate is found in the dark-adapted, light-adapted, or M states of bacteriorhodopsin.
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页码:8366 / 8371
页数:6
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