Bacteriorhodopsin (BR), a model system in biotechnology, is a G-protein dependent trans membrane protein which serves as a light driven proton pump in the cell membrane of Halobacterium salinarum. Due to the linkage of retinal to the protein, it seems colored and has numbers of versatile properties. As in vitro culture of the Halobacteria is very difficult, and isolation is time consuming and usually inefficient, production of genetically modified constructs of the protein is essential. There are three important characteristics based on protein catalytic cycle and molecular functions of photo-electric, photochromic and proton transporting, which makes this protein as a strategic molecule with potential applications in biotechnology. Such applications include protein films, used in artificial retinal implants, light modulators, three-dimensional optical memories, color photochromic sensors, photochromic and electrochromic papers and ink, biological camouflage and photo detectors for biodefense and non-defense purposes.
Bacteriorhodopsin (BR) mutagenesis plays an important role in the development of BR-based materials and tools with enhanced optical and electrical properties. Previously reported protocols for generating BR mutations are inefficient for the preparation and purification of mutant proteins. Therefore, a series of BR mutations were generated by using improved methods, which are described in further detail. The functional activity of the recombinant proteins was confirmed by spectroscopic and electrochemical assays. Modified proteins with different wavelengths and activities form a foundation for color-sensitive sensors and can be utilized to produce unique bioelectrical and biotechnological tools and materials. The proton-pumping activity of the generated mutant D85E was normal, indicating that the mutant could be used in light batteries. However, mutants D85Q and D85N were almost inactive; and D85N had a prolonged M state, suggesting that it could be utilized in light memories.
The properties of bacteriorhodopsin (BR) can be manipulated by genetic engineering. Therefore, by the methods of gene engineering, Asp85 was replaced individually by two other amino acids (D85V, D85S). The resulting recombinant proteins were assembled into soybean vesicles retinylated to form functional BR-like nano-particles. Proton translocation was almost completely abrogated by the mutant D85S, while the D85V mutant was partially active in pumping protons. Compared with wild type, maximum absorption of the mutants, D85V and D85S, were 563 and 609 nm, which illustrated 5 nm reductions (blue shift) and 41 nm increases (red shift), respectively. Since proton transport activity and spectroscopic activities of the mutants are different, a wide variety of membrane bioreactors (MBr) have been developed. Modified proteins can be utilized to produce unique photo/Electro-chromic materials and tools.
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