Structure of the N intermediate of bacteriorhodopsin revealed by x-ray diffraction.

Abstract

X-ray diffraction experiments revealed the structure of the N photointermediate of bacteriorhodopsin. Since the retinal Schiff base is reprotonated from Asp-96 during the M to N transition in the photocycle, and Asp-96 is reprotonated during the lifetime of the N intermediate, or immediately after, N is a key intermediate for understanding the light-driven proton pump. The N intermediate accumulates in large amounts during continuous illumination of the F171C mutant at pH 7 and 5°C. Small but significant changes of the structure were detected in the x-ray diffraction profile under these conditions. The changes were reversible and reproducible. The difference Fourier map indicates that the major change occurs near helix F. The observed diffraction changes between N and the original state were essentially identical to the diffraction changes reported for the M intermediate of the D96N mutant of bacteriorhodopsin. Thus, we find that the protein conformations of the M and N intermediates of the photocycle are essentially the same, in spite of the fact that in M the Schiff base is unprotonated and in N it is protonated. The observed structural change near helix F will increase access of the Schiff base and Asp-96 to the cytoplasmic surface and facilitate the proton transfer events that begin with the decay of the M state.The active site of an ion pump must communicate alternately with the two opposite sides of the membrane. Change of the protein conformation, linked to this switch, is therefore expected to be an essential step in the reaction cycle. In the light-driven proton pump bacteriorhodopsin (bR), the switch must occur after proton transfer toward the extracellular side but before proton transfer from the cytoplasmic side-i.e., while the proton binding site, the retinal Schiff base, is unprotonated. Thus, the switch reaction is expected to take place during the lifetime of the M intermediate in what was termed the Ml to M2 reaction (1-5). Structural changes are indeed revealed by neutron, x-ray, and electron diffraction when photostationary states are created where the M intermediate accumulates (6-9). We have shown that this structural change is closely related to the deprotonation of Schiff base; in the original structure (conformation E) the proton channel is open to the extracellular side, and when an M-type conformation is assumed (conformation C) it is open to the cytoplasmic side (10). Thus, the proton transport mechanism is elegantly explained by the alternating protein conformation model (11, 12). Light energy is utilized to cause the initial proton transfer that triggers the structural transition from conformation E to conformation C, and that results in the change of access of the retinal Schiff base as well as further changes in proton affinity (pK) of donor and acceptor groups so as to drive proton transfers at strategic locations in the protein.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" i...

Propagating Structural Perturbation Inside Bacteriorhodopsin: Crystal Structures of the M State and the D96A and T46V Mutants

“…The disorder along helix F begins at Val-177, which is displaced in a manner consistent with the outward tilt of the cytoplasmic end of helix F detected in many other studies (16)(17)(18)(19)(20)(21)(22). Thus, it may be the result of the tilt that disrupts the crystal locally, and as such constitutes evidence for this largescale conformational shift.…”

Electron diffraction studies of light-induced conformational changes in the Leu-93 → Ala bacteriorhodopsin mutant

“…Our observations support the conclusion from spin-labeling experiments (27) that there are likely to be conformational changes at the cytoplasmic end of helix C well after the formation of the M intermediate. However, because changes are observed in helices B, C, F, and G, it is clear that the structural changes on helices B and G are not likely to be limited to only the first phase of the conformational switch (13).…”

A local electrostatic change is the cause of the large-scale protein conformation shift in bacteriorhodopsin

“…Maps of the changed structure of the M state obtained by diffraction have been interpreted as density increases at the cytoplasmic halves of helices B and G. When M is stabilized under various conditions this is seen to be followed by tilting of the cytoplasmic end of helix F away from the central cavity (14)(15)(16). The latter change persists in the N state stabilized in mutants (17,18). A component of these large-scale structural changes, termed collectively the E to C shift (4), may be the basis of the extracellular-tocytoplasmic change of the connectivity of the Schiff base, the postulated ''reprotonation switch'' (2-4).…”