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Insights into the perception of light

Rhodopsin mediates between the visual world and our brain. Whether or not these proteins are able to turn light into electrical impulses depends very much on the bonds between the individual molecule groups. A team of biophysicists led by Dr. Reiner Vogel at the University of Freiburg have used infrared spectroscopy to gain detailed insights into the rhodopsin centre. They have discovered how the molecular side chains of the proteins have to interact in order for the brain to “understand” the visual environment.

The world as we see it is perceived through specialised sensory cells in the retina (rods and cones), which translate information about the environment, i.e. light energy, into electrical impulses. But how is this information transduced to the brain? Different wavelengths of light are absorbed by sensory cells where they come into contact with rhodopsin molecules – proteins that are stored in the discs of rod cells and consist of opsin and retinal, a photoreactive chromophore, in the central pocket. Activated retinal looks like a bent leg. When light is absorbed, retinal stretches its “knee”, thereby displacing the different side chains of the enveloping protein. This induces a conformation change in rhodopsin that activates the associated G protein and triggers a biochemical cascade. This leads to the closure of the ion channels in the sensory cell membrane, thereby changing the current flow. The cells of the optic nerve register the resulting voltage change and transmit this information to higher brain areas.

Rhodopsin is an important model

“The rhodopsin proteins of the human eye are highly efficient,” said Dr. Reiner Vogel from the Institute of Molecular Medicine and Cell Research at the University of Freiburg. “Two to three light quanta are sufficient to activate rhodopsin and transmit information about the environment to the brain.” Vogel and his two colleagues in the biophysics work group are also investigating other receptors that interact with G proteins (so-called G protein-coupled receptors, GPCRs for short), which transduce extracellular stimuli into the cell. This also involves receptors that are activated through neurotransmitters or hormones rather than through light, for example the adrenalin receptor in the heart muscle. Rhodopsins are particularly interesting for the biophysicists because they are well known and are excellent models for other GPCRs. For example, they are very sensitive to light, which enables the researchers to activate rhodopsin with short laser pulses and observe clearly defined temporal courses in the reactivity.
The schematic shows three types of G protein-coupled receptors (GPCRs) that are activated in different ways (light, ligands, signalling proteins). The protein on the left represents rhodopsin with the retinal molecule in its central pocket (Figure: Dr. Reiner Vogel)
Over the last few years, Vogel and his team have been trying to find out the rhodopsin regions that are set in motion as a response to a light pulse, thus inducing a conformational change in the protein, thereby activating it. This happens when the membrane-bound receptor domains (so-called α-helices) move against each other. This breaks up the existing bonds between the side chains of the α-helices and new bonds form in different places. The researchers are able to measure this process using infrared spectroscopy. In contrast to visible light, infrared light is relatively long-wave. The wavelengths, which are introduced into a sample through a beam, are absorbed by molecule bonds, which can be visualised in an absorption spectrum of the light emitted from the sample. The individual peaks of such a spectrum correspond to different bond types, for example ionic bonds between the charged side chains of amino acids.
Model of a rhodopsin receptor in a biomembrane; three important bond types are highlighted (Figure: Dr. Reiner Vogel)

Looking for important bonds

“The spectrum of inactive rhodopsin has different peaks which reflect the different types of bonds of a molecule,” said Vogel. “If this spectrum is subtracted from the spectrum generated by photoactivated rhodopsin, then this produces the bond peaks that differentiate the inactive from the active form of rhodopsin.” These bonds break and form again when rhodopsin detects light. Vogel and his colleagues have since discovered two types of bonds, including one that is located right in the centre of rhodopsin and must be broken in order for the receptor to refold upon the absorption of light and interact with G protein.

Vogel and his colleagues are using these methods to investigate rhodopsin and other GPCRs in their native environment of biomembranes. The physiological conditions are key for the proper functioning of the receptors, however the investigation of rhodopsins in their natural environment requires specifically optimised methods (see box). Vogel and his colleagues are also able to transfer their knowledge about rhodopsin to other GPCRs, for example to the neurotransmitter receptors in the brain or muscles. Such receptors are of pharmacological importance, because they do not function properly in certain diseases.
Surface spectroscopy
Principle of optimised surface spectroscopy: The receptor monolayer in the biomembrane (top), the connecting proteins (in the centre) and the gold particle layer (bottom). The crystal through which the light is guided, would, in this schematic, be located below the gold layer. (Figure: Dr. Reiner Vogel)
Optimised surface spectroscopy:
Dr. Reiner Vogel and his colleagues are investigating receptor proteins in natural membranes under physiological conditions, which requires the sample medium to have optimal temperature, pH value and salt concentrations. However, the water in the medium absorbs a lot of infrared light and makes measurement with infrared beams difficult. That is why the Freiburg scientists work with biomembrane receptor monolayers. These thin layers are applied to crystals through which the infrared light is guided. The light does not then enter the sample and is therefore not absorbed by the water. Quantum physical phenomena nevertheless ensure that the infrared light interacts with the sample. This interaction alters the beam emitted from the crystal and can be measured. However, the signal of such a thin biomembrane receptor layer is very weak. Therefore, the biophysicists apply a specifically structured gold film between the crystal and the biomembrane receptor layer. The receptors are coupled to the gold particles using specific proteins. The bonds between the gold atoms have special characteristics and enhance the signal.
mn – 11 December 2008
© BIOPRO Baden-Württemberg GmbH
Further information:
Dr. Reiner Vogel
Institute of Molecular Medicine and Cell Research
University of Freiburg
Hermann-Herder-Str. 9
79104 Freiburg, Germany

Tel.: +49 (0)761 203-5391 (office)
Tel.: +49 (0)761 203-5395 (laboratory)
Fax: +49 (0)761 203-5390
Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/insights-into-the-perception-of-light