Genetic Engineering of Bacteriorhodopsin (The Purple Power Protein) for improving its applications

Dr. Rajesh Thakur Assistant Professor
Department of Bio & Nano Technology, Guru Jambheshwar University of Science & Technology, Hissar, Haryana – 125001. India.

rthakur99@rediffmail.com

• Bacteriorhodopsin (bR) is a light-activated proton pump that is formed when the 248 amino acid protein Bacterio-opsin is coupled to the chromophore all-trans retinal.

Bacteriorhodopsin is a Dynamic, Transmembrane Protein
Several transmembrane spanning helices 3 Tyr residues hold the BR in place

Retinal sits at the junction separating the two “half” channels Notice the Bacteriorhodopsin is surrounded by Asp and Arg

Photons for Protons
• Bacteriorhodopsin takes energy from photons • This energy is utilized to create a proton gradient by pumping protons outside the cell. • Protons are allowed back into the cell by an ATP synthase. • In a nutshell: Photons are used to power the cell
hv H+ H+

+++++++ --------ADP

F0F1 ATPase

ATP

Source of BR
• BR is primarily found in Archaebacteria Halobacteria salinarium • These are halophilic bacteria (i.e., they are found in very salty water e.g. Great Salt Lake)

Occurrence of Halobacteria
• Genera Halobacteria belonging to the halophilic archaea group are the only genera that contain retinal proteins. • Halophilic archaea group in general grow on organic substrates and respire in a standard fashion using oxygen. However, the high salt environment in which the Halobacteria occur, allow only 1/5th availability of oxygen. • Under the anaerobic conditions that commonly occur in their habitats, these organisms have acquired alternative means for energy conversion. • This system does not use chlorophyll-based reaction centers but rather relies on retinal as a photon absorber and retinal-containing proteins as energy transducers.

• Halobacteria have developed a set of four retinal proteins,
– Rhodopsin – Halorhodopsin - Bacteriorhodopsin - Sensory rhodopsin

• Two of them have sensory function,

• While other two convert light energy to chemical energy.

• Bacteriorhodopsin is also found in some unicellular plants and some fungi. • Evolution has optimized this protein for high photochemical efficiency, thermal stability and cyclicity, as the organism must be able to function in a hot, stagnant and resource-limited environment.

• It absorbs light @ 570 nm (visible green light)
• Red and Blue light is reflected, giving membrane its purple colour

Biotechnological Production of BR
• •
i.

BR forms 2D crystals in vivo, and these can be isolated as purple membranes. The isolation is facilitated by the following:
Halobacterial cells are unstable in water and cell lysis occurs. ii. The cell membrane is fragmented, and the crystalline patches of the purple membrane are set free as fragments of largest size and highest buoyant density. These two specific features are used in the isolation procedure, either in a combination of sedimentation and isopycnic gradient centrifugation leading to a product of highest purity, or by a filtration procedure.

Technical Applications of BR
• In most of the applications, bacteriorhodopsin is used in the Purple Membrane (PM) form, because its liberation from the crystalline package reduces both the chemical and thermodynamic stability. The three basic molecular properties which may be used in technical applications are:
– – – Photoelectric property Photochromic property Proton transport property

Photoelectric Property
• • A single PM sheet contains several thousands of unidirectionally oriented BR molecules. Upon illumination, a photovoltage up to 250mV per single bacteriorhodopsin layer is generated, and this may be used either as an indicator or control element for various applications. Upon absorption of a photon, the bacteriorhodopsin molecule undergoes a series of very rapid molecular changes, one of which is the generation of a photovoltage. The photovoltage generated can be easily measured by embedding the PM layer between two transparent electrodes. A voltage is induced only during the light intensity change. The polarity of the signal is different for the OFF  ON and the ON  OFF transition. This is called ‘Differential Responsivity’ of PM layers.

• •

Photoelectric Properties
Electroni c interface are a Electronic interface area
Light sensitive pixelated area

In a pixelated structure which is coated with oriented bacteriorhodopsin, photovoltages are measured only in those spots where a change in the light intensity occurs.
This is called novelty filtering.

Movement of object

Differential response of sensor array (novelty detection)

Artificial Retina
A hybrid semiconductor bacteriorhodopsinbased artificial retina is fast and inexpensive and mimics the motion sensitivity of the human retina.

Photochromic Properties
• During the photocycle, bacteriorhodopsin cycles through a pair of spectroscopically distinguishable intermediates, which have an absorption maximum which is different from that of the initial B state.
In most applications the photochromism of bacteriorhodopsin is used in connection with the purple to yellow absorption change which is related to M state formation. Upon acidification, a blue membrane is formed which has a significantly different photocycle. The formation of a 9-cis retinal-containing state is observed, and this is thermally stable. This opens the route to long-term storage materials based on bacteriorhodopsin.

Photocycle
Purple • • The energy trapping conformational transition occurs within one ps Deprotonation and reprotonation steps are characterized by intermediates which contain distinct absorption spectra One full turn of the cycle occurs within a ms Hence each step has a characteristic colour Every sec, 50 protons are exported

• • • Yellow

Applications based on the photochromism
• Photochromic color classifier • Photochromic / Electrochromic inks • Photochromic photographic film • Applications in Biosensors • Long-term photorewriteable storage of information

• Neural networks
• 3-D information storage • Nonlinear optical filtering • Holographic pattern recognition and interferometry

Some Other Applications
 Cheap, easy way of accumulating protons • Industry • Fuel cell cars

 Switches
 Y-Channels  Artificial retina

 3-D Memories
 Electro-optically controlled spatial light modulators

Suitability of Bacteriorhodopsin
Bacteriorhodopsin has been studied over the past two decades as a material for technical applications because:

• Its stability is adequate
• It has several technically interesting functions • Tools for both its modification and production in technical quantities have been developed • Stable and standard strains of bacteria are available for its production • It offers various interface principles, whether optical, electrical, or chemical. However, the native protein still lacks the overall efficiency necessary for commercial viability and virtually all successful photonic devices using bacteriorhodopsin are based on chemical or genetic variants of the native protein.

Short lifetime of optical products
• Optical films incorporating BR in a polymeric matrix have already been demonstrated by Ames researchers and others, to allow the recording of extremely high resolution (>5000 lines/mm) holograms with reasonable diffraction efficiency, that are optically erasable and can be re-written millions of times without film or material degradation.

• However, images or holograms recorded at room temperature in the natural form of BR (called wildtype BR) have a short lifetime (on the order of milliseconds to a few seconds) due to thermal relaxation of the excited molecular state back down to the ground molecular state.
• While appropriate for some optical processing applications, this is unacceptable for data storage applications, where the data must remain permanently recorded until actively erased.

BR gene and polypeptide
• 786 nucleotide structural gene
• 13 AA precursor sequence +248 AA in mature BR +1 AA at C-terminal sequence • No intervening sequences • It is a 26 KD protein

Are There Any Highly-conserved Residues?
L. Brown, 2001 • Results of BLASTing the H. salinarium BR showed very high homology among all BR from a number of different Halobacteria. • Around the schiff base at position 216, there is no deviation in AA composition for a good 4.5 Angstroms. • This type of analysis shows that the entire retinal binding pocket is highly conserved. Therefore, many of the AAs in BR are structurally and/or catalytically important.

Site directed mutagenesis
• BR readily lends itself to genetic engineering, which allows the generation of genetic variants that may possess significantly different optical characteristics. In one example of genetically engineered BR, a variant known as D85N is created by substituting an asparagine residue for an aspartic acid at position 85 in the 248 amino acid polypeptide chain near the retinal part of the molecule.

This variant shows dramatically different behavior in the sense that the excited molecular state formed by photon absorption in the red part of the visible spectrum is thermally stable and does not decay back down to the ground state.
However, it is still optically erasable via the absorption of a blue photon, and is thus a good preliminary candidate for use in holographic data storage

Semi random mutagenesis and directed evolution
• Semi-random mutagenesis and directed evolution will play a prominent role in future efforts in bioelectronic optimization. • Various strategies under study are focused upon modulating the photoelectric behavior of bR. • Successful alterations can lead to modified bR that respond differently to changes in pH, hence allowing control on its photoelectric behavior.

Quantum Mechanics studies
• Quantum mechanics-molecular modeling study of the BR molecule imparts a deeper understanding of changes it undergoes during the photocycle. • This will enable evaluation of the effects of specific genetic mutations on the photocycle and to identify genetic mutations that will modify the photocycle in a controlled fashion. • Being able to simulate the molecular behavior of BR in different genetic configurations will allow us to guide the genetic engineering changes to be performed experimentally in the laboratory.

Conclusion
• Genetic engineering approaches have already been applied to BR as a means to alter the molecule's optical properties, primarily through the technique of site-specific mutagenesis.

• It is hoped that the thermal relaxation will be blocked in the genetically engineered BR without compromising the attractive photochemical properties that enable various promising applications.

References
• • • F. T. Hong and F. H. Hong (1992) AIP Conf. Proc. / Volume 262 / Issue 1. 262, pp. 204-217 Wise KJ, Gillespie NB, Stuart JA, Krebs MP, Birge RR. (2002) Trends Biotechnol. 2002 Sep;20(9):387-94. Hampp NA. (2000) Appl Microbiol Biotechnol. 2000 Jun;53(6):633-9.


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Oesterhelt, D. (1998) The structure and mechanism of the family of retinal proteins from halophilic archaea. Curr. Opin. Struct. Biol. 8, 489–500.

Lanyi, J. K. (1998) Understanding structure and function in the light-driven proton pump bacteriorhodopsin. J. Struct. Biol. 124, 164–178.

Thanks for your Attention
Dr. Rajesh Thakur rthakur99@rediffmail.com

Assistant Professor Department of Bio & Nano Technology, Guru Jambheshwar University of Science & Technology, Hissar, Haryana – 125001. India.

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