A step closer to image capture using biological photoreceptors

Light capture in biology is all around us, but putting this to good use technologically has proved challenging. Professor Anthony Watts and Olivia Berthoumieu, together with collaborators in Oxford, have brought this ambition a bit closer, as reported in four recently published papers (1-4) and as a result of an eight year effort.

The red/purple colour of salt beds comes from the patches in the outer membrane of salt-loving micro-organism Halobacterium salinarum, in which the coloured light-receptor protein bacteriorhodopsin is embedded

The red/purple colour of salt beds comes from the patches in the outer membrane of salt-loving micro-organism Halobacterium salinarum, in which the coloured light-receptor protein bacteriorhodopsin is embedded (Click to enlarge)

‘Coupling biomolecules into nanotechnological devices is a major hurdle,’ says Tony Watts, ‘but by strategically engineering a specific amino acid into a robust bacterial photoreceptor, and coupling this to a gold electrode, we have been able to detect molecular photoswitching in an unprecedented way.’

The bacterial photoreceptor bacteriorhodopsin has long been the focus of potential biotechnological development. Under low oxygen conditions, it uses sunlight to generate an electrical potential across the outer membrane of the Archae in which it is found (Halobacterium salinarum). That then acts as the energy source to drive metabolism, live and reproduce.

Bacteriorhodopsin is responsible for the red-purple colour of marine salt-reclamation beds. The natural purple membranes in which the photoreceptor protein normally exists can be produced in the laboratory from H.salinarum. These membranes have long been recognised as the best environment to enable possible technological development for light or energy capture, and for memory storage (it has been suggested that proteins like bacteriorhodopsin can store more than 20 times the information of the same amount of conventional magnetic recording material).

But progress has been slow in realising the aims of achieving energy capture using these membranes. Previous efforts to immobilise bacteriorhodopsin have been plagued with difficulties. Foremost amongst these is the difficulty of achieving good electrical coupling to an electrode. It has also not been possible to immobilise the membrane to an electrode asymmetrically, so that the electrical generation can flow in the same direction.

A high-resolution image of specially prepared bacteriorhodopsin immobilised on a gold surface through a strategically engineered linking group in the protein, which is around 4nm (4 billionths of a metre) across

A high-resolution image of specially prepared bacteriorhodopsin immobilised on a gold surface through a strategically engineered linking group in the protein, which is around 4nm (4 billionths of a metre) across (Click to enlarge)

Now, by introducing two major molecular modifications to the membranes and receptor protein, these difficulties have been overcome and a highly responsive light capture configuration has been achieved.

The molecular modifications have led to removal of most (75%) of the highly charged lipids from the bacterial membranes whilst retaining light-response ability. In addition, a cysteine amino acid has been strategically engineered into a highly responsive and well-defined location on only one side of bacteriorhodopsin.

The immobilisation of the receptor on the gold electrode is now closer than for membranes, as well as being asymmetric, ensuring that the electrical current generation by light all goes in one direction. The problem of low response when using symmetrically immobilised unmodified bacterial membranes has been overcome, with the closer intimate coupling enhancing the signal many times when compared with unmodified membranes.

The group detected photon switching in this new receptor configuration using electrically-coupled atomic force microscopy methods that can intimately address the photoreceptor at the molecular level.

A fine tip is brought gently into contact with the immobilised photoreceptor protein to detect electrical current and voltage that switch in response to light

A fine tip is brought gently into contact with the immobilised photoreceptor protein to detect electrical current and voltage that switch in response to light (Click to enlarge)

At the heart of light capture in bacteriorhodopsin is retinal. Retinals act as nanoswitches. When light hits, they can move protons as charged electrical particles, generating life-sustaining electrical potential, as for bacteriorhodopsin in the outer membrane of H.salinarum. Or they can alter the conformation of the photoreceptor to initiate a signal amplification cascade – a process that takes place in the eyes of higher organisms.

Retinal is a relatively recent product of evolution. It has only been used in living systems for about 400 million years. By contrast, green chlorophylls and related chromophoric molecules have been used by cyanobacteria, and subsequently plants, for 3 – 3.5 billion years.

Light detection in biological systems is very different from in man-made detectors. In nearly all current light detector devices based on silicon technology, such as cameras and image detection devices, the light is either detected or not. Colour images need to be produced through colour filters to deconvolute the image, and the final image is then reconstructed.

In biological molecules such as bacteriorhopsin, the detector is colour sensitive across a range of wavelengths, including into the far infra-red or ultra violet - extremes in the spectrum not visible by humans, but visible to some animals such as spiders (far UV), cats (far IR) and deep-sea organisms (at very low light levels).

The sensitivity of retinals to different wavelengths is dependent upon the environment of the protein in which they are embedded. By exploiting this phenomenon, the wavelength of the light to which a photoreceptor responds can be ‘tuned’ through directed evolution. In bacteria, this can be achieved by growing under different coloured light.

For bio-nanotechnological use, photoreceptor proteins have many advantages over man-made silicon technology based light and energy capture devices. Importantly, the biological detectors are about 10,000 times smaller (at about 4x10-9 m in diameter). They have much higher resolution, and respond much faster (in 10-12 - 10-15 seconds) and at much greater efficiency (60-70% of the light is captured) than any man-made silicon devices.

Although man-made single photon detection devices are available, NASA and military research organisations are making very significant investments in designing better imaging devices. But these still cannot match biology in its efficiency of photon detection which is many orders of magnitude better in sensitivity or response times than top-down designed devices.

‘There is still a long way to go, but we do need to learn from nature,’ says Tony Watts. ‘Although this is of some possible future technological benefit, we need to understand at a more fundamental level how light and energy is captured in biology. The kind of experimental set-up we have developed will allow us now to explore in a much more controlled environment how this is achieved. We will be able to probe the molecular mechanisms and very complex photophysics that nature has evolved and exploited in such an exquisite way to enable life to exist on the earth.'

'We have developed a basic image detection and energy capture device based on a robust biological photoreceptor. Although the dreams of solar energy generation so often mentioned in the literature are unlikely to be achieved due to the low currents being captured (pA), colour-sensitive image detection at super-high resolution - not at megapixels but terapixel resolution - is a step closer'.

References

  1. Berthoumieu, O., Patil, A., Wang, X., Aslimovska, L., Jason, D., and Watts, A. (2011) Engineered bacteriorhodopsin: a molecular scale conductance photoswitch Eur. Biophys. J., 40:pS235
  2. Berthoumieu, O., Patil, A., Wang, X., Aslimovska, L., Jason, D., and Watts, A. (2012) Molecular scale conductance photoswitching in engineered bacteriorhodopsin, Nano Letters, 12, 899–903. doi: 10.1021/nl203965w
  3. Patil, A. V., Premaruban, T., Berthoumieu, O., Watts, A., and Davis, J. J. (2012) Enhanced photocurrent in engineered bacteriorhodopsin monolayer films. J. Phys. Chem. B,116, 683–689.doi: 10.1021/jp210520k
  4. Patil, A. V., Premaruban, T., Berthoumieu, O., Watts, A., and Davis, J. J. (2012) Engineered Bacteriorhodopsin: A Molecular Scale Potential Switch. Chemistry – A European Journal (in press)





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