residues: Asp-96, Asp-85, Asp-212, and Arg-82 play critical roles in the
proton transfer. Later electron-microscopy studies of rhodopsin showed
that the structures of the two proteins are very similar.
Although achievement of the structure provided a detailed molecular
model of the function, a number of questions were still unanswered. The
spacing of the amino acid residues was too far apart to provide a uninter-
rupted proton pathway, and the light-induced structural changes were not
determined. Answers to these questions required structural studies that
provided more detail of not only the dark state but also the light-induced
intermediate states.
The quality of the structure was improved after well-ordered, three-
dimensional crystals of bacteriorhodopsin were obtained after many years
of effort by different groups. The breakthrough was the inclusion of lipids
in the crystallization solutions. The lipid concentration was poised so that
phase separation occurred, with the protein being concentrated in the cubic
phase along with the lipid (see Chapter 4). The protein is solubilized with
lipids rather than detergents and was thus incorporated into the cubic lipid
phase where the regular arrangement of the lipids promotes the forma-
tion of protein crystals.
The resulting structure determined from the crystals confirmed the ear-
lier electron-microscopy results with the only major differences found in
the loop regions, which were difficult to model in the electron micrographs.
Because the crystal structure was determined at a much higher resolution,
with the best crystals reaching a resolution limit of 1.55 Å, structural
features were now resolvable (Figure 17.9). In particular, the structure
showed the presence of water molecules bound inside the protein. The
water molecules answered the question of how the proton could traverse
the long 10-Å distances found between the amino acid residues forming
the proton pathway. The pathway has many water molecules that pro-
vide the 2-Å steps needed for individual proton transfers.
The light-induced structural changes were identified by trapping the
protein in different photochemical states and determining the struc-
tures in those states. The very early state shows only small changes near
the retinal. The latter state shows not only the isomerization of the
retinal but also the changes in the positions of the waters and amino
acid residues forming the proton pathway. In particular, the bound
water molecules near the trans-retinal are in different positions after the
cis-retinal is formed, which is consistent with a movement of a proton
along the pathway.
Because these structural states can be related directly to the spectro-
scopic states, the photochemical cycle of bacteriorhodopsin has been well
established (Figure 17.10). Because the retinal is tethered, the isomeriza-
tion leads to changes in the positions of the surrounding helices, which are
relatively small – less than 1 Å – but sufficient to drive the proton transfer.
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