Researchers at the University of Bonn and the Caesar Research Center have succeeded in freezing proteins ultra-fast after a precisely defined period of time. They were able to track structural changes on a microsecond scale and with sub-nanometer precision. Due to its high spatial and temporal resolution, the method allows monitoring of rapid structural changes in enzymes and nucleic acids. The results are published in the Journal of the American Chemical Society.
If you want to know what the spatial structure of a biomolecule looks like, you have a formidable arsenal of tools. The most popular are electron microscopy and X-ray diffraction, which can reveal even the smallest details of a protein. However, a significant limitation of these methods is that they generally provide static images, which are often insufficient to understand biomolecular processes in precise mechanistic terms. Therefore, a long-term goal of many research groups around the world has been to follow the movements within a macromolecule such as a protein over time as it does its job, like in a movie. The research groups led by Prof. Olav Schiemann from the Institute for Physical and Theoretical Chemistry at the University of Bonn and Prof. Dr. but.
They chose an ion channel for their investigation. It is a protein that forms tiny pores in the cell membrane that are permeable to charged particles called ions. “This chain is normally closed,” explains Schiemann. “It only opens when a cellular messenger, called cAMP, binds to it. We wanted to know exactly how this process works.”
Mini magnets for measuring distances
To do this, the researchers first mixed the channel protein and cAMP, then quickly frozen the solution. In the frozen state, the protein structure can now be analyzed. In order for their method to work, they had attached molecular electromagnets to two points of the channel. The distance between these magnets can be determined with an accuracy of a few angstroms (ten billionths of a millimeter) using a sophisticated method called PELDOR, which works like a molecular ruler. In recent years, the method has been considerably refined and improved in Schiemann’s group.
“However, this only gives us a static picture of cAMP binding to the ion channel,” says Schiemann. “So we repeated the freezing process at different times after mixing the two molecules. This made it possible to reconstruct the movements of the protein after cAMP binding – like a film, which is also made up of of a sequence of images. “
At the center of this procedure is a sophisticated method that allows samples to be mixed and frozen very quickly at a specific time. The technique, called “microsecond hyperquenching” (abbreviated MHQ), was originally developed at the University of Delft, but later fell into disuse. It was decisively rediscovered and refined by Kaupp’s group.
“In the MHQ device, the cAMP molecule and the ion channel are mixed at an ultra-fast rate,” Kaupp explains. “Then the mixture is projected in the form of a very fine jet onto a very cold metal cylinder at -190 ° C, which rotates 7,000 times per minute. It was particularly difficult to transfer the frozen samples for the PELDOR measurement from the metal plate to a thin plate. glass tubes, and keep them frozen while waiting. We had to design and manufacture special tools for this. “
Freezing in 82 millionths of a second
The entire mixing and freezing process takes just 82 microseconds (one microsecond is one millionth of a second). “This allows us to visualize very rapid changes in the spatial structure of proteins,” explains Tobias Hett, one of the two doctoral students who contributed significantly to the success. The advantage of the method is its combination of high spatial and temporal resolution. “This represents a major advance in the study of dynamic processes in biomolecules,” Kaupp points out.
The researchers now plan to use their method to take a closer look at other biomolecules. They hope to gain new knowledge, for example on how enzymes and nucleic acids work. The importance of this information is best illustrated by the recent global wave of structural research on the SARS coronavirus-2: the so-called spike protein of the virus also undergoes a structural change when human cells are infected. Clarification of this mechanism will provide valuable information on how to target the mechanism of infection with new drugs.
Sample preparation, experimental execution and data analysis are very complex. The results of the study therefore also reflect successful scientific cooperation with researchers led by Prof. Helmut Grubmüller from the Max Planck Institute for Biophysical Chemistry in Göttingen and Prof. Dr. Heinz-Jürgen Steinhoff from the University of Osnabrück.
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