The role of water in the extracellular half channel of bacteriorhodopsin.
The changes in the photocycle of the wild type and several mutant bacteriorhodopsin (D96N, E204Q, and D212N) were studied on dried samples, at relative humidities of 100% and 50%. Samples were prepared from suspensions at pH approximately 5 and at pH approximately 9. Intermediate M with unprotonated Schiff base was observed at the lower humidity, even in the case where the photocycle in suspension did not contain this intermediate (mutant D212N, high pH). The photocycle of the dried sample stopped at intermediate M1 in the extracellular conformation; conformation change, switching the accessibility of the Schiff base to the cytoplasmic side, and proton transport did not occur. The photocycle decayed slowly by dissipating the absorbed energy of the photon, and the protein returned to its initial bacteriorhodopsin state, through several M1-like substates. These substates presumably reflect different paths of the proton back to the Schiff base, as a consequence of the bacteriorhodopsin adopting different conformations by stiffening on dehydration. All intermediates requiring conformational change were hindered in the dried form. The concentration of intermediate L, which appears after isomerization of the retinal from all-trans to 13-cis, during local relaxation of the protein, was unusually low in dried samples. The lack of intermediates N and O demonstrated that the M state did not undergo a change from the extracellular to the cytoplasmic conformation (M1 to M2 transition), as already indicated by Fourier transform infrared spectroscopy, quasielastic incoherent neutron scattering, and electric signal measurements described in the literature.
Ganea
C
C
Department of Biophysics, Carol Davila Medical University, Bucharest, Romania.
Gergely
C
C
Ludmann
K
K
Váró
G
G
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019617:Halobacterium salinarum / Q000737:chemistry; D006813:Humidity; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D012545:Schiff Bases / Q000737:chemistry; D013053:Spectrophotometry; D014867:Water / Q000378:metabolism*
D007473:Ion Channels; D012545:Schiff Bases; D014867:Water; D001436:Bacteriorhodopsins