THE ROLE OF WATER IN THE EXTRACELLULAR HALF CHANNEL OF BACTERIORHODOPSIN

The changes in the photocycle of the wild type and several mutant bact eriorhodopsin (D96N, E204Q, and D212N) were studied on dried samples, at relative humidities of 100% and 50%. Samples were prepared from sus pensions at pH similar to 5 and at pH similar to 9. Intermediate M wit h unprotonated Schiff base was observed at the lower humidity, even in the case where the photocycle in suspension did not contain this inte rmediate (mutant D212N, high pH). The photocycle of the dried sample s topped at intermediate M-1 in the extracellular conformation; conforma tion change, switching the accessibility of the Schiff base to the cyt oplasmic side, and proton transport did not occur. The photocycle deca yed slowly by dissipating the absorbed energy of the photon, and the p rotein returned to its initial bacteriorhodopsin state, through severa l M-1-like substates. These substates presumably reflect different pat hs of the proton back to the Schiff base, as a consequence of the bact eriorhodopsin adopting different conformations by stiffening on dehydr ation. 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 l ocal relaxation of the protein, was unusually low in dried samples. Th e lack of intermediates N and O demonstrated that the M state did not undergo a change from the extracellular to the cytoplasmic conformatio n (M-1 to M-2 transition), as already indicated by Fourier transform i nfrared spectroscopy, quasielastic incoherent neutron scattering, and electric signal measurements described in the literature.