Molecular energy machine as a movie star

Researchers at the Paul Scherrer Institute, PSI, have used the Swiss Light Source SLS to record a molecular energy machine in action and thus reveal how energy production works at the level of cell membranes. To this end, they have developed a new method of investigation that could make the analysis of cellular processes much more efficient than before. They have now published their findings in the journal Science.

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

PSI researchers have already been able to elucidate part of this process with free electron X-ray lasers (FELs) such as SwissFEL. Now they have also managed to record the still unknown part of the process in some kind of molecular film. For this, they chose a method that was previously only usable in the FEL and then developed it for use in the Swiss Light Source SLS. The study highlights the synergy between the analytical options of these two large-scale PSI research centers. "With the new method at SLS, we can now follow the last part of the bacteriorhodopsin movement, where the steps are in the order of a millisecond," says Tobias Weinert, first author of the article. "With the measurements made at the FELs in the US and Japan, we had already measured the first two sub-processes before SwissFEL was commissioned," says Weinert. "These are happening very quickly, in the space of a few microseconds, between femtoseconds." A femtosecond is equivalent to one billionth 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, the proteins are transformed into crystals. A laser beam imitating sunlight triggers the movement sequence in the protein. X-rays that then reach the sample generate diffraction images, which are recorded by a high-resolution detector. From these, computers generate an image of the structure of the protein at every moment.

The film created from measurements at the SLS shows how the structure of the bacteriorhodopsin molecule changes over the next 200 milliseconds after its activation by light. With that, a so-called "photocycle" of the molecule has now been elucidated.

Bacteriorhodopsin works 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 surface, there are more protons than on its inner side. The cell uses this gradient to gain energy in its metabolism by allowing protons located elsewhere to balance different internal and external concentrations. In doing so, the cell produces ATP, a source of universal energy in living beings. Subsequently, bacteriorhodopsin restores the concentration gradient.

"In the new study, we were now able to see the biggest structural changes in real time of a molecule." By "big", the scientist means nine angstroms, 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 molecules of water forms, responsible for the transport of the proton through the cell membrane. "Before us, no one had ever observed this chain of water directly," notes the biochemist happily.

These observations were made possible only by the modification of the method previously used by SwissFEL for use by SLS and the new high-speed and fast Eiger detector at SLS. Weinert is convinced that the new investigation method using synchrotrons like SLS will inspire research worldwide. "Researchers can use the new method and become much more efficient, because there are many more synchrotrons in the world than free electron lasers, and you need fewer protein crystals than it can." This is needed for experiments with FELs, "adds Weinert.

However, for very fast molecular processes, as well as to obtain particularly sharp images and precise results, the researchers rely on SwissFEL. "The processes at the beginning of the photocycle take place in a few seconds, and it is possible to observe only such rapid chemical reactions at the level of the FELs." In addition, structures can be recorded with higher resolution at the LEF. Because so many photons hit the sample at a time at the linear accelerator, the detector can capture an extremely sharp image.

Weinert stresses the synergy between the two large-scale research facilities: "SwissFEL has only a small amount of beam time, thanks to the measurements made by SLS we can ensure the success of the project of our experience at SwissFEL. "

The researchers have now published the results of the study in the journal Science.