Scale Conformational Changes in the M, N, and O Intermediates

F through the hydrogen bond of the C=0 of Ala-215 to water 501 and from there to the indole N of Trp-182 is lost. 

In the O-like state the extracellular ends of helices A, B, C, and D are tilted outward, but their cytoplasmic ends are not displaced. Helix E is tilted also, but around a pivot point near its middle, so its extracellular and cytoplasmic ends are displaced outward and inward, respectively. If this structure is indeed like that of the O state, the implication is that the protein undergoes a scissoring motion in the second half of the photocycle. It begins with a splaying of the cytoplasmic side of the seven helical bundle in M, which continues in N but reverses in O and opens the extracellular cavity instead. These suggested large-scale global motions are in sharp contrast with the relatively small (1-2 A) and more local atomic displacements in the first half of the photocycle. The rationale must be that the structure of the protein in the unilluminated state predisposes it to the early reactions in the cycle, but the later reactions require drastically different conformations. 

In the second half of the photocycle the Schiff base is reprotonated in a protonation equilibrium with Asp-96, and this is followed by reprotonation of Asp-96 from the cytoplasmic surface. Unlike in the extracellular region, where a three-dimensional hydrogen-bonded network already exists to facilitate the earlier events, involving transfer of protons 

At pH 8 the kinetic relationship of M, N, and O is simpler than at lower pH and can be described with the scheme M -o- N — O. The protonation equilibrium of the Schiff base with Asp-96, the M - N reaction, is pH independent consistent with internal proton transfer, but the reprotonation of Asp-96, the N —O reaction, is pH dependent consistent with proton uptake from the bulk. Thus, one should expect a two-stage conformational shift, to enable ion transfer first between the Schiff base and Asp-96, and then between Asp-96 and the cytoplasmic surface. 

The M to N and the N to O reactions both occur on the millisecond time scale, yet Asp-96 cannot be in communication with the cytoplasmic surface at the time its protonation equilibrium with the Schiff base is established. It is likely that this is ensured by the hydrophobic shield between Asp-96 and the aqueous interface that contains the side chains of Phe-42, Leu-100, Phe-171, and Leu-223. At the time Asp-96 is repro-tonated from the bulk in the N to O reaction, this shield must be made permeable, while the pathway between Asp-96 and the Schiff base is abolished. The crystal structure of the O-like state suggests how these two goals are accomplished (Fig. 6, see color insert Rouhani et al., 2001). 

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