Bacteriorhodopsin

In a cell, be it prokaryotic or eukaryotic, there are plenty of functions to perform. Most of which are carried out by proteins. Depending on the location, proteins can be soluble or transmembrane or amphitropic. The functions of transmembrane proteins range from signal transduction, ion channels, energy transduction, structural role to even cell recognition, among others. If one protein that can be considered as a perfect model for studying transmembrane proteins, it has to be Bacteriorhodopsin also known as BR. It has been widely studied for more than three decades now and thereby has also laid the basis for existing knowledge on transmembrane proteins.

Bacteriorhodopsin is a seven-helix transmembrane protein, produced by and Archaebacteria called Halobacterium salinarum. This microbe is found in salt mines and salt lakes, to be precise, it is found in high salt concentration and warm habitats. This halophilic, obligate aerobe is capable of producing energy in low oxygen concentration and under illumination, through a different pathway instead of using the electronic respiratory chain. With the help of this alternative pathway cells are rendered the capability of surviving but not growing. Bacteriorhodopsin transforms light energy into electrochemical energy and when coupled with an ATP synthase, light energy is transformed into chemical energy. Thus, this complex is said to be the simplest photosynthetic system known. BR contains a covalently linked molecule of retinal which responds to photon stimuli. After the light stimulus, retinal isomerizes give rise to some conformational changes in the protein, which creates a proton gradient from the cytoplasm to external medium. This proton gradient is used by the ATP synthase to produce ATP. BR is an integral membrane protein which is usually found in two dimensional crystalline patches known as “purple membrane” which may occupy up to 50% of the surface area of the archaeal cell. BR represents the 75% (w/w) of the purple membrane, while the other 25% consists of a mixture of ether-linked lipids as squalene, sulphate glycolipids and phosphatydiglycerolphosphates. The BR forms repeating elements arranged in chains. Each chain consists of seven transmembrane alpha helices and contains one molecule of retinal buried deep within, the typical structure for retinylidene proteins. The BR molecule is purple and is most efficient at absorbing green light at wavelength 500-560nm, with maximum absorption at 568nm. Bacteriorhodopsin belongs to a family of bacterial proteins related to vertebrate rhodopsin, the pigments that sense light in the retina. Many molecules have homology to BR, including some directly light activated channels like channelrhodopsin and the light driven chloride pump halorhodopsin. All other phototrophic systems in bacteria, plants and algae use bacteriochlorophylls or chlorophylls rather than bacteriorhodopsin. These organisms also produce a proton gradient but in different and more indirect way by involving an electron transfer chain consisting of several other proteins. It must also be noted that chlorophylls are aided in capturing light energy by other pigments known as antennas which are not present in bacteriorhodopsin-based systems. Also, chlorophyll based phototrophy is coupled to carbon fixation i.e. the incorporation of carbon dioxide into larger organic molecules which is again not true for bacteriorhodopsin-based system.

BR consists of an apoprotein which is bacterioopsin, also called as BO. It is a protein encoded by the bop gene in the H. salinarum genome, consisting of 248 amino acids with a molecular weight of 26 KDa. Tertiary structure consists of seven transmembrane α-helices, named from A to G, which are connected by loops at each side of the membrane. Retinal remains surrounded by the helices in the middle of the protein, determining its two halves, the cytoplasmic and the extracellular one. Retinal is covalently linked to the apoprotein via a protonated Schiff base with Lys216, giving rise to a chromoprotein which is called BR. Three molecules of BR are grouped. These molecules form a trimer, then form a hexagonal network of hexamers, which eventually assumes the lattice of a 2D crystal.

Retinal – Retinal is the chromophore of the protein. It is a carotene derivative of vitamin A and it contains a β-ionone ring. As mentioned earlier, it is covalently linked to the protein at the Lys216 through a protonated Schiff Base. In its free form, retinal presents an absorbance maximum of 380nm dissolved in ethanol. In the resting state of the protein, that is in the unphotolyzed state, there is an equilibrium between two forms, all-trans and 13-cis with a ratio of 1:2, respectively. The absorbance maximum of this chromoprotein is 558nm, and it represents the so-called dark-adapted form. When this dark-adapted BR is illuminated, the equilibrium moves completely to a ratio of a 100% of all-trans retinal, yielding the light adapted form with an absorbance maximum of 568nm. The retinal is mainly surrounded by hydrophobic and aromatic side chains, enclosing retinal in the proper place, which is called as the Retinal Binding Pocket. Most of the retinal surrounding residues are aromatic amino acids such as Trp (86, 138, 182, 189) and Tyr (57, 83, 185). This sort of a bulky chain seems to be necessary to place correctly the retinal and avoid steric wrong conformations. Besides these aromatic residues, there are also some polar and hydrophilic (Thr90, Asp212, Asp85) and hydrophobic residues (Leu93) in the retinal binding pocket. Hydrophobic residues such as Trp and Leu and the barely reactive Met seem to carry out a structural role whereas more hydrophilic residues such as Asp, Thr, and Tyr seem to perform a more functional role. Residues such as Thr90 or Pro186, which have no relation with the proton pathway, could be considered as relevant candidates for the transmission of conformational changes from the retinal to the protein, assuming a dynamic role. Asp85, Asp212 and Arg82 are not included in the Retinal binding pocket, but form part of the counter ion cloud, which stabilizes the positive charge of the Schiff Base. Some buried water molecules are also induced in the counter ion.