Biological light sensor filmed in action

The molecule retinal is a form of vitamin A and is of central importance to humans, animals, certain algae, and many bacteria. In the retina of the human eye, retinal triggers the process of vision when it changes its shape under the influence of light. In a similar form, certain bacteria also use this reaction to pump protons or ions through the cell membrane. Light energy can be stored in this way, as in the reservoir of an alpine hydropower plant, so that it is available on demand as biological fuel. To ensure efficient utilisation of light, the retinal molecule is embedded in proteins that play a critical role in regulating the process. The protein-regulated reaction of retinal is one of the fastest biological processes and occurs within 500 femtoseconds (a femtosecond is one-millionth of one-billionth of a second). That is roughly a trillion times faster than the blink of an eye, says Jörg Standfuss, who heads the group for time-resolved crystallography in the Division of Biology and Chemistry at PSI. What happens in the process on the atomic level has now been captured for the first time by PSI researchers, in 20 snapshots that they have assembled into a molecular movie. No one has previously measured a retinal protein at such high speed and with such precision. It's a world record, says Jörg Standfuss, who led the study.

The film shows the transition between the main states of retinal within the first picoseconds after activation in the binding pocket of the bacteriorhodopsin. (Video: Paul Scherrer Institute/Przemyslaw Nogly and Tobias Weinert)

The researchers studied the protein bacteriorhodopsin, which is found in simple microbes. When the retinal molecule embedded in the bacteriorhodopsin traps a light particle, it changes its original elongated shape into a curving form, like when a cat arches its back, explains the PSI researcher. Such changes can also be observed when retinal is examined in a solution without protein. There, though, different reactions, which are also less productive, take place. Proteins are like factories in which chemical reactions run especially efficiently, Jörg Standfuss explains. We wanted to look at how this interplay between the protein and the molecule functions.

A surprising observation

The researchers discovered that water molecules in the vicinity of the retinal play a critical role. They were able to observe how the water molecules moved aside and made room for the retinal molecule to do its cat-arching-its-back move – in the technical jargon, a trans-cis isomerisation. This detail, which no one had seen before, surprised Jörg Standfuss, as he explains with the help of the cat analogy: You expect that a cat might arch its back to scare another one away. But here the second cat runs away even before the first has arched its back. Computer simulations confirm the measurements, which could be explained by ultrafast quantum processes.

Besides the retinal reaction, the researchers were also able to detect protein quakes that had been predicted by theory. The arching of the cat's back does not require the entire energy of the light that falls on the protein. Excess energy is released, evidently, not in the form of heat but rather in vibrations of the protein.

New measurements planned at SwissFEL

For their images, the PSI researchers traveled to California, to the free-electron X-ray laser LCLS at Stanford University. In the future, they will be able to realise such films right at PSI with the newly commissioned facility SwissFEL. For such studies, the sample is illuminated with extremely short and intense flashes of laser-quality X-ray light. The X-ray beams are diverted in different directions by the sample and generate diffraction patterns from which the original structure can be calculated.

As samples, the researchers use tiny crystals in which the bacteriorhodopsin is densely packed in an ordered state. The light sensor in the bacteriorhodopsin is excited by a short pulse from an optical laser. Afterwards, the X-ray flash hits the crystal and lights up the scene. The time between the optical signal and the X-ray flash determines how far the reaction will have progressed. Individual snapshots taken at different points in time can be spliced together into a movie.

In serial crystallography, crystals are injected into an X-ray beam. When the beam and the crystal meet, rays of light are diffracted. The diffracted light rays are recorded by a detector. From the light patterns that many identical crystals produce at the detector, the structure of the crystals can be determined. For time-resolved experiments, an additional optical laser is used to activate the biomolecules in the crystal at a definite point in time. (Video: Paul Scherrer Institute/Mahir Dzambegovic)

After studying bacteriorhodopsin, the PSI researchers want to use SwissFEL to investigate the retinal in rhodopsin in our eyes. Similar retinal proteins can also be artificially incorporated into nerve cells, so it becomes possible to selectively activate nerve cells with light and study their function. With these retinal proteins, one can activate any region in the brain with the help of light, says Jörg Standfuss, explaining the goal of the new field called optogenetics. Measurements with SwissFEL are expected to contribute to the improvement of optogenetics applications.

Text: Barbara Vonarburg

About PSI

The Paul Scherrer Institute PSI develops, builds and operates large, complex research facilities and makes them available to the national and international research community. The institute's own key research priorities are in the fields of matter and materials, energy and environment and human health. PSI is committed to the training of future generations. Therefore about one quarter of our staff are post-docs, post-graduates or apprentices. Altogether PSI employs 2100 people, thus being the largest research institute in Switzerland. The annual budget amounts to approximately CHF 390 million. PSI is part of the ETH Domain, with the other members being the two Swiss Federal Institutes of Technology, ETH Zurich and EPFL Lausanne, as well as Eawag (Swiss Federal Institute of Aquatic Science and Technology), Empa (Swiss Federal Laboratories for Materials Science and Technology) and WSL (Swiss Federal Institute for Forest, Snow and Landscape Research).

(Last updated in May 2018)