Mechanism of proton pumping in bacteriorhodopsin by solid-state NMR: the protonation state of tyrosine in the light-adapted and M states.
Solid-state 13C 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'-13C]Tyr-labeled bR, regenerated with [14-13C]retinal as an internal marker to identify the photocycle states. The [14-13C]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'-13C 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.(ABSTRACT TRUNCATED AT 250 WORDS)
McDermott
A E
AE
Francis Bitter National Magnet Laboratory, Massachusetts Institute of Technology, Cambridge 02139.
Thompson
L K
LK
Winkel
C
C
Farrar
M R
MR
Pelletier
S
S
Lugtenburg
J
J
Herzfeld
J
J
Griffin
R G
RG
D001436:Bacteriorhodopsins / Q000737:chemistry*; D003624:Darkness; D006217:Halobacterium / Q000032:analysis; D007700:Kinetics; D008027:Light; D009682:Magnetic Resonance Spectroscopy; D045322:Photosynthetic Reaction Center Complex Proteins / Q000378:metabolism; D011522:Protons; D012545:Schiff Bases; D014443:Tyrosine / Q000737:chemistry*
D045322:Photosynthetic Reaction Center Complex Proteins; D011522:Protons; D012545:Schiff Bases; D014443:Tyrosine; D001436:Bacteriorhodopsins