Protein conformational changes during the bacteriorhodopsin photocycle. A Fourier transform infrared/resonance Raman study of the alkaline form of the mutant Asp-85-->Asn.
Bacteriorhodopsin is a light-driven proton pump, which undergoes a photocycle consisting of several distinct intermediates. Previous studies have established that the M-->N step of this photocycle involves a major conformational change of membrane embedded alpha-helices. In order to further investigate this conformational change, we have studied the photocycle of the high pH form of the mutant Asp-85-->Asn (D85Nalk). In contrast to wild type bacteriorhodopsin, D85Nalk has a deprotonated Schiff base and a blue-shifted absorption near 410 nm, yet it still transports protons in the same direction as wild type bacteriorhodopsin (Tittor, J., Schweiger, U., Oesterhelt, D. and Bamberg, E. (1994) Biophys. J., 67, 1682-1690). Resonance Raman spectroscopy of D85Nalk and D85Nalk regenerated with retinal labeled at the C-15 position with deuterium reveals the existence of an all-trans configuration of the chromophore. Fourier transform infrared difference spectroscopy shows that the photocycle of this light-adapted form involves similar events as the wild type bacteriorhodopsin photocycle including the M-->N protein conformational change. These results help to explain the ability of D85Nalk to transport protons and demonstrate that the M-->N conformational change can occur even in the photocycle of an unprotonated Schiff base form of bacteriorhodopsin.
Nilsson
A
A
Physics Department, Boston University, Massachusetts 02215, USA.
Rath
P
P
Olejnik
J
J
Coleman
M
M
Rothschild
K J
KJ
D001216:Asparagine; D001224:Aspartic Acid; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism*; D019617:Halobacterium salinarum / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D017354:Point Mutation; D011487:Protein Conformation; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013059:Spectrum Analysis, Raman / Q000379:methods; D013816:Thermodynamics
D001224:Aspartic Acid; D001436:Bacteriorhodopsins; D001216:Asparagine