The gaseous protein is cautiously placed on graphene
However, to image single proteins with electron holography, the Swiss researchers still needed a carrier material for the proteins that is transparent to electron waves as well as a method to place biomolecules on it without causing damage. Graphene proved to be the most suitable material for the carrier. Researchers at the Max Planck Institute for Solid State Research found the best solution for depositing proteins on the sheets made up of carbon layers: electrospray ion beam deposition, which was developed by a team headed by Stephan Rauschenbach in Klaus Kern’s department. The researchers expose the protein solution to a high electrical voltage so that the liquid is highly charged. Electrical repulsion then causes the liquid to atomize into a fine mist. When the mist droplets are exposed to a vacuum, the liquid evaporates and the dissolved constituents, i.e. proteins and impurities, remain behind as gases. A mass spectrometer then sorts the proteins according to their mass-to-charge ratios and also separates out impurities.
“Our method makes it possible to transfer single biological molecules into the vacuum and deposit them on a surface so gently that their fragile three-dimensional folded protein structure is preserved,” says Stephan Rauschenbach. “Thanks to preparative mass spectrometry, we also prevent contamination of the graphene samples with other molecules, which is crucial for the quality of the holographic image.” Mass spectrometry also makes it possible to separate protein mixtures or pure proteins from complexes with binding partners.
Information on the assembly of subunits
Once Stephan Rauschenbach and his colleagues have deposited the proteins on the graphene substrates in Stuttgart, the samples have to be transported to Zurich, where the electron holographic microscope is located. The samples must arrive in an uncontaminated state, meaning that no other molecules can be allowed to settle on the graphene. To transport the samples to Switzerland, the researchers have developed a case in which an ultra-high vacuum prevails, as in the apparatus itself.
Thanks not least to the meticulous care and cleanliness observed during the preparation and transport of the samples, electron holograms already achieve a resolution of less than one nanometre. “This allows us to investigate how the individual subunits of large protein complexes are assembled,” Stephan Rauschenbach says. The first holograms of single proteins also provide information about their three-dimensional structure.
“However, to accurately image protein structures at the atomic level, we still have to improve the resolution somewhat,” explains Klaus Kern. “, there are no physical obstacles preventing this.” The Zurich- and Stuttgart-based scientists now plan to construct a microscope in which the vibrations of proteins are suppressed by cooling the samples to around minus 200 degrees Celsius. In addition, a unique precision laboratory has recently been constructed at the Max Planck Institute in Stuttgart, which offers perfect conditions for highly sensitive measurements such as holography. This laboratory was built on the initiative of Klaus Kern and is currently the gold standard for a low-vibration measuring environment. As soon as the electron holography microscope has been optimized, biomedical scientists can use this new instrument to study the intricacies of how the tools of life function.
Prof. Dr. Hans-Werner Fink
Tel. 0041 44 635 58 01