Bacteriorhodopsin Experiment

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Bacteriorhodopsin is located in the plasma membrane of Halobacterium halobium, where it acts as a proton-pump, transporting protons out of the cell. Since its discovery in 1979, much research has been done on Bacteriorhodopsin and there have been many advancements in our knowledge. Firstly, on its structure and function and secondly, on the conformational changes it undergoes during retinal isomerisation.

Bacteriorhodopsin is a globular protein, (Henderson, 1975) which acts as a light driven proton- pump located in the native purple membrane of Halobacterium halobium. It was first discovered by Stoechenius and his lab in 1979 (Stoeckenius, 1979). Although Henderson and Unwin had already made a three-dimensional map of
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As Bacteriorhodopsin is the only protein component of the purple membrane, we can assume that any protein referred to in their reviews was Bacteriorhodopsin. They discovered 3 identical units rotated by 120o. Each unit has seven closely packed alpha helical segments, with each alpha helix containing 20-25 predominantly hydrophobic residues with one covalently attached retinal molecule. (White, 1994). The seven transmembrane α-helices are typical of all G-protein coupled receptors. (Lanyi, 1998) The retinal molecule is covalently attached to Lys-216 on helix G by a protonated Schiff base. The location and positioning of the retinal group was discovered using medium-resolution diffraction studies (Jubb, 1984). In the middle of the α-helices, there is a hydrophobic pocket, which lies approximately 15Å from the extracellular surface and this is where the retinal is found (Bonting, 1981). The retinal molecule is anti-parallel to the membrane plane and perpendicular to the α-helices (Henderson, 1975). The retinal itself is a long unsaturated hydrocarbon chain, which is known as the aldehyde form of Vitamin A. The residues responsible for transporting the protons are also contained within this interhelical domain where the retinal is bound (Lanyi, 1998). In the ground state this central channel is …show more content…
This is known as the deprotonated form and from this state the retinal has the ability to return to the all-trans state, while another proton transfer occurs (Bonting, 1981). The second proton transfer also occurs in the extracellular half-channel (Hans-Thomas Richter, 1996). Asp-85 is deprotonated by protonating the empty proton release site (Lanyi, 1998).The proton is transferred from the protonated residue to the Schiff base, causing the salt-bridge to reform (Bonting, 1981). Proton uptake from the cytoplasm uses Asp-96 as the proton donor. This uptake is regulated by protein conformation and depends on this region being hydrated (Lanyi, 1998) and reprotonation of Asp-96 coincides with the retinal returning to the all-trans state (Lanyi, 2005).
The translocation of the proton must be close to the two aqueous interfaces as bound water has a very important role to play in the movement of the proton in the mechanism of proton translocation as proton transfer occurs via a bridging water molecule (Lanyi, 1998). This proton translocation is unidirectional and the proton is always moved from the cytoplasm to the extracellular surface. This is because the Schiff bas is not always accessible from both half- channels. The proton affinity for Asp-85 and Asp-96 also changes, depending on which stage of the photocycle is occurring (Lanyi,

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