Bacteriorhodopsin
Created by: Michael Terango
Bacteriorhodopsin (1DZE) found in Halobacterium salinarum is a light-driven proton pump. The light is detected by a single retinal molecule bound to the last of seven transmembrane helices in the protein by a protonated Schiff base linkage (1). When the retinal molecule absorbs visible light, it undergoes a conformational change through five intermediate stages (K, L, M, N, and O intermediates) of a photocycle (2). These protein conformational changes cause a proton to translocate from the cytosolic area into the extracellular area across the purple membrane of Halobacterium salinarum (7). Archaea, particularly Halobacteria, use the resulting proton gradient formed by bacteriorhodopsin to create chemical energy through ATP synthesis (1). Scientists study the bacteriorhodopsin proton pump as a model for many ion pumps in cells. According to the Expasy database, the molecular weight of bacteriorhodopsin is 26,800.58 Da, and the isoelectric point is 3.67 (5).
The protein only has one subunit, a polypeptide polymer, but that is broken down into seven helices each twenty nonpolar residues long (11). All of the seven main alpha helices are connected to each other. The grooves found on the protein’s surface allows for the hydrophobic lipid tails to interact with it while still maintaining a hydrophilic center to allow proton movement (9). The retinal contained in the G helix powers the proton pump through visible light absorption that isomerizes the molecule and causes shifts in the conformation of the entire protein based on its central location in bacteriorhodopsin (1). The central location of the retinal contributes to the protein’s purpose of establishing a proton gradient within the membrane.
Many residues found in the sequence of bacteriorhodopsin contribute to the photocycle of the protein in the proton pump. Located in the middle of helix G of bacteriorhodopsin, Lys-216 is the site of the Schiff base linkage, and it is susceptible to X-ray radiation (2). Asp-85 is the primary acceptor of a proton from the Schiff base during the formation of the M intermediate (8). The hydrophobic interaction between Val-49 and Leu-93 keep the Schiff base from the acidic cytosol (9). Asp-96 donates a proton to the Schiff base from the hydrophobic section of the cytosolic membrane when the bacteriorhodopsion conforms from an M intermediate to an N intermediate (1). The cytoplasmic membrane surface donates a proton back to Asp-96 to be used again in the cycle (9).
The secondary structure of the M intermediate of bacteriorhodopsin is 66% helical (9 helices) and 4% consisting of beta sheets (2 strands) (1). The other 30% of the M intermediate of bacteriorhodopsin consists of random coils, ligands, and water molecules that all contribute the function of bacteriorhodopsin (1). Six hydrogen bonds stabilize the twisted anti-parallel beta sheet in the B-C interhelical loop, which provides the protein structural integrity. The isomerization of retinal allows for direct movement in the cytoplasmic end of the F helix (6). The alpha helices of bacteriorhodopsin have specified charged polar sides to carry the proton through the protein while still remaining in the membrane.
The M intermediate of bacteriorhodopsin does not have associated substrates or ions, but the protein is surrounded by water molecules that contribute to the photocycle. A water molecule displaces the G helix of bacteriorhodopsin by hydrogen bonding to the space between Lys-216 and the rest of the peptide skeleton during formation of the M intermediate (1). Water stabilizes Asp-85 in the M intermediate through hydrogen bonding. This conformational change causes enough strain to distort the loop between the F and G helices, which further disrupts hydrogen bonds between Glu-194 and Glu-204 and the salt bridge at Glu-204 and Arg-82 (6). These changes are coupled with the displacement of the G helix through the M intermediate formation, showing that the protein’s association with water molecules directly affects its function as a proton pump (1).
The molecular formation of the tertiary structure allows for the protein to function as a single monomeric protein. Trimers of bacteriorhodopsin form a hexagonal lattice in its crystallized form (9). An intermolecular salt bridge forms between Lys-40 and Asp-104 that form the tight trimeric unit of bacteriorhodopsin through helix to helix interaction (9). Nineteen side-chain to side-chain or side-chain to main-chain hydrogen bonds between neighboring helices keep the barrel like structure of bacteriorhodopsin (6). This ring provides a tunnel across the purple membrane of Halobacterium salinarum through which the proton may exit. Bacteriorhodopsin clusters are found within the purple membrane in repeating arrays for order.
Bacteriorhodopsin contains many ligands in its structure that contribute to the proton pump and stability of the protein within the membrane. Retinal is the chromophore that contributes to the photocycle by undergoing isomerization from the trans to the cis configuration (1). Beta-D galactose is an isomer of glucose that helps polysaccharide uptake in glucose hydrolysis as an attachment to the membrane (1). Other polysaccharide ligands including alpha-D glucose, alpha-D mannose, and 3-phsphoryl-[1,2-di-phytanyl]glycerol provide hydrogen bonds to specific residues on the protein to hold it in place (1).
Although there are no drugs in development that utilize bacteriorhodopsin, the function of the protein in Archaea provides information on structural methods of ion pumps found in the membranes of bacterial and eukaryotic cells. Bacteriorhodpsin is a homologue to many sensory rhodopsins found in eukaryotes, including Acetabularia rhodopsin (3AM6) found in the marine plant Acetabularia acetabulum (10). The Dali server is a database of proteins that contrasts tertiary structures through a calculation of the differences in tertiary distances and gives a Z-score, which shows the closeness to a given protein (3). Acetacularia rhodopsin has a Z-score of 28.9, and because the Z-score is above 2, the tertiary structures of bacteriorhodopsin and acetacularia rhodopsin are very similar (3). Using the BLAST database, which determines the primary structure similarity between two proteins reveal an E score that defines how statistically close a sequence is to another (4). The E score of the acetacularia rhodopsin is 2e-12, and since it is much smaller than 1, they are primarily of the same sequence (4). According to the Expasy database, the molecular weight of the acetacularia rhodopsin is 25,546.26 Da, which is slightly smaller than bacteriorhodopsin (5). The isoelctric point of acetacularia rhodopsin is 5.10 (5). Like bacteriorhodopsin, acetacularia rhodopsin contains retinal, forms a ring of alpha helices, and acts as a proton pump (10). Opposite to bacteriorhodopsin, acetacularia rhodopsin intakes a proton before the protein releases a proton because of the lack of a functional proton-releasing group of residues or the difference in pKa of the proton-releasing residue into the extracellular region (10). The binding site of acetacularia rhodopsin is different than the binding site found in bacteriorhodopsin. This difference causes acetacularia rhodopsin’s inability to absorb transitional light (10). The comparison between bacteriorhodopsin and rhodopsin found in eukaryotic cells identify the structural functions between the proton pumps.