Researchers have used the Swiss Light Source SLS to record a molecular energy machine in action and thus to reveal how energy production at cell membranes works. For this purpose they developed a new investigative method that could make the analysis of cellular processes significantly more effective than before. They have now published their results in the journal Science.

In all living things, structural changes in proteins are responsible for many biochemically controlled functions, for example energy production at cell membranes. The protein bacteriorhodopsin occurs in microorganisms that live on the surface of lakes, streams, and other bodies of water. Activated by sunlight, this molecule pumps positively charged particles, protons, from the inside to the outside through the cell membrane. While doing this, it is constantly changing its structure.

The researchers were already able to elucidate one part of this process at free-electron X-ray lasers (FELs) such as SwissFEL. Now they have also managed to record the still unknown part of the process in a kind of molecular movie. "With the new method at SLS, we can now follow the last part of the movement of bacteriorhodopsin, where the steps are in the millisecond range", explains first author of the paper. "With measurements at FELs in the USA and Japan, we had already measured the first two sub-processes before SwissFEL was commissioned", the author says. "These take place very fast, within femtoseconds to microseconds." A femtosecond is one-trillionth of a second.

To be able to observe such processes, the researchers use so-called "pump-probe" crystallography. With this method, they can take snapshots of protein movements that can then be assembled into movies. For the experiments, proteins are brought into crystal form. A laser beam, imitating sunlight, triggers the sequence of movements in the protein. X-rays that hit the sample afterwards produce diffraction images, which are recorded by a high-resolution detector. From these, computers generate an image of the protein structure at each point in time.

The movie created from the measurements at SLS shows how the structure of the bacteriorhodopsin molecule changes in the next 200 milliseconds after it is activated by light. With that, a complete so-called "photocycle" of the molecule has now been elucidated.

Bacteriorhodopsin functions as a biological machine that pumps protons from inside the cell through the membrane to the outside. This creates a concentration gradient at the cell membrane. On its outer side, there are more protons than on its inner side. The cell uses this gradient to gain energy for its metabolism by allowing protons elsewhere to balance out the externally and internally different concentrations. In doing so, the cell produces ATP, a universal energy source in living things. Subsequently, bacteriorhodopsin restores the concentration gradient.

"In the new study, we were now able to see the largest real-time structural changes in a molecule ever" - by "large" the scientist means nine angstroms, that is, one-millionth of the thickness of a human hair. Through these structural changes, a gap opens up in the protein in which a chain of water molecules forms, and this is responsible for the proton transport through the cell membrane. "Before us, no one had ever observed this water chain directly", the biochemist notes happily.

"Researchers can use the new method and become much more efficient, since worldwide there are many more synchrotrons than free-electron lasers. Besides that, you need fewer protein crystals than are required for experiments at FELs", the author adds.

https://www.psi.ch/en/node/29378?access-token=9atQMPm6kZ3mwZTY

https://science.sciencemag.org/content/365/6448/61