Cells produce a large number of different protein complexes, each made up of many individual proteins. These protein complexes, like ribosomes for example, are what regulate almost all of the vital biological functions of a cell.
Biologists have been successful in determining the structure of many of these complexes, but so far there is less research on how individual proteins assemble and then change over time. Conventional approaches have so far proved insufficient to study the exact course of these reactions in cells, in particular when it comes to large complexes.
A group of ETH researchers led by Karsten Weis and research associate Evgeny Onischenko at the Institute of Biochemistry at ETH Zurich are now presenting a new approach. Their method makes it possible to follow the dynamics of complex protein assemblies, even for very large ones, with high temporal resolution. The study has just been published in the journal Cell.
Inspired by metabolic analysis
ETH researchers call their new approach KARMA, which stands for kinetic analysis of incorporation rates in macromolecular assemblies and is based on methods of studying metabolic processes. Scientists who study metabolism have a long history of using radioactive carbon in their work, such as labeling glucose molecules, which cells absorb and then metabolize. Radioactive labeling allows researchers to track where and when glucose molecules or their metabolites appear.
“This type of research inspired us to apply a similar principle to explore the reactions that occur in the assembly of protein complexes,” Weis explains. In their approach, ETH researchers work with labeled amino acids, the building blocks of proteins, which contain heavier carbon and nitrogen isotopes. In a culture of yeast cells, the team replaces the light amino acids with their heavier counterparts. Yeast uses these heavy amino acids in protein synthesis, which changes the molecular weight of all newly produced proteins.
A timescale for assembling a complex
To isolate the protein complexes, the researchers remove the yeast cells from cultures at regular intervals and use mass spectrometry to measure the tiny difference in weight between molecules with heavier amino acids and those without. This indicates the age of a protein in a complex. Basically, the older the protein, the earlier it was incorporated into the complex. Based on these age differences, researchers apply kinetic state models to ultimately reconstruct the precise assembly sequence of a given protein complex.
As a case study to validate their method, Weis and his team chose the nuclear pore complex in yeast cells. This structure comprises between 500 and 1000 elements composed of about 30 different proteins, each in several copies, which makes it one of the largest protein complexes known.
Thanks to KARMA, biochemists at ETH were able to obtain a detailed map of the modules integrated into the structure and when. One of their discoveries was a hierarchical principle: individual proteins form subunits in a very short time, which then assemble from the center to the periphery in a specific sequence.
“We have shown for the first time that some proteins are used very quickly in the assembly of the pore complex, while others are not incorporated until after about an hour. It’s an incredibly long duration, ”says Weis. A yeast cell divides every 90 minutes, which means it would take almost an entire generation to complete the assembly of this vital pore complex. It is not clear exactly why the assembly of new pores takes so long compared to the yeast reproductive cycle.
ETH researchers also show that once the pore assembly is complete, the parts of the complex are very stable and durable – in the interior scaffolding, for example, hardly any components are replaced during its life. . In contrast, proteins at the periphery of the nuclear pore complex are frequently replaced.
Defective nuclear pores facilitate disease
Nuclear pores are among the most important protein complexes in cells, as they are responsible for the exchange of substances and molecules between the cell nucleus and the cytoplasm. For example, they transport messenger RNA from the nucleus to the cellular machinery outside the nucleus, which needs these molecules as blueprints for new proteins.
In addition, nuclear pores play direct and indirect roles in human diseases. As a result, changes in the nuclear pore and its proteins can impact the development of conditions such as leukemia, diabetes, or neurodegenerative diseases such as Alzheimer’s disease. “In general, however, the reasons why pore defects cause these types of diseases are not well understood,” Weis explains, explaining that KARMA may help to better understand these problems in the future.
“Although we applied KARMA to a single protein complex in this study, we are excited about its future applications. Our method will now allow us to decipher the sequence of a whole series of biological processes, ”says Weis. Their technique can be used, for example, to study molecular events that occur during the infection cycle of viruses such as COVID-19 and potentially help find new drug candidates that break that cycle.
The new method can also be applied to other biological molecules besides proteins, such as RNA or lipids.
Onischenko E, Noor E, Fischer JS, Gillet L, Wojtynek M, Vallotton P, Weis K: kinetics of maturation of a multiprotein complex revealed by metabolic labeling, Cell, Available online December 16, 2020. DOI: 10.1016 / j.cell.2020.11.001
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