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Next generation single molecule protein fluorescence

Periodic Reporting for period 4 - SMPFv2.0 (Next generation single molecule protein fluorescence)

Reporting period: 2019-05-01 to 2021-04-30

In more complex organisms like humans, very dynamic proteins, so called intrinsically disordered proteins (IDPs) enriched. Those proteins are most famous for their involvement in misfolding neurodegenerative aging diseases like Alzheimer, Parkinson, Huntington. However, they are actually key to many important and precise cellular mechanisms, likely because the ability to encode multiple tasks into those dynamic polymer like proteins vs more stable proteins apparently outweighs the seeming disadvantages. This was likely key to evolution of more complex species. Studying such highly dynamics is a major challenge and requires novel technology development. The ability to observe single molecules using fluorescence was awarded in 2014 with the Nobel Prize in Chemistry, as it enables a direct look on how fundamental molecular processes work in biology. This ultimately gives an unbiased view at how the molecular building blocks of live are designed by nature, and how they function together. Furthermore, malfunctions in such processes can be directly visualized using modern spectroscopy and microscopy techniques, which is of high relevance for health and society.
Despite all progress, single molecule fluorescence science suffers from the need for site-specific labelling and the intrinsic low throughput. In this project, we aimed to improve single molecule science, by integrating chemical biology and microfluidic tools into a synergistic process on how to study disordered biological building blocks with high resolution. The tools were directly used to study a set of IDPs highly relevant to a fundamental function of the eukaryotic cell, which in turn also feedbacks on the tool development.
In the eukaryotic cell all genetic material is stored in the nucleus, which for example protects it modifications due to pathogens, like viruses. However, constant information exchange in the form of biomolecules entering and exiting the nucleus is essential for cellular function. Intrinsically disordered proteins termed nucleoporins form a permeability barrier of still elusive nature inside nuclear pore complexes spanning the nuclear envelope. While key to controlling nucleocytoplasmic transport, likely due to their disordered nature, many of those proteins can also take on vital functions in processes like gene regulation elsewhere in the cell. The fundamental principles behind such multifunctionality are largely unknown.
We follow a seeing is believing approach. However, to visualize a molecular process, fluorescence probes need to be incorporated into the protein of choice without altering protein function. The smaller the probe, and the more site-specifically it can be introduced, the more information can be extracted about the system.
Genetic Code expansion (GCE), is a technology that enables to swap a normal amino acid, against a noncanonical amino acid harboring a unique functionality to which we can couple small fluorescent dyes. This way we can introduce very small stable fluorophores into proteins that we want to study. This protein engineering is done inside the cell, and this can have an impact on cellular physiology and lead to unwanted effects. In a series of publications (most prominent Nikic et al Angewandte 2016 and Reinkemeier Science 2019) we showed that we can visualize single proteins inside nuclear pore complexes labelled with single dyes in a site-specific manner. However, as powerful as GCE is, it has also many shortcomings that limit its application in cellular biology since it does also interfere with canonical function of the cell. However, we have developed a conceptually new approach how to solve this problem, which I detail in the next section.
Furthermore, and to cope with the small throughput of our fluorescent microscopy approaches, we combined our technologies also with a novel microfluidic flow platform, in which proteins pass one after the other through an observation area (JCB 2019). Doing this enabled us to identify that nucleoporins can form a simple liquid droplet like aggregate/condensate that already obeys many characteristics of functional nuclear pore complexes. Our work provided a new model platform to study the nuclear transport machinery under controlled conditions and already enabled us new insights into the key features that charaterize functional nuclear transport.
Eukaryotic cells achieve higher complexity by minimizing crosstalk between different process by dedicating specific functions to different organelles. While some of those organelles are membrane encapsulated like the nucleus itself, more recently it has become clear that also some organelles, like stress granules, the nucleoli etc. are formed by phase separation, which leads to very high local concentration of biomolecules like RNA and protein. Inspired by this natural concept, we thought to develop a new organelle, inside a eukaryotic cell that is dedicated to protein engineering. More precisely, due to the interdisciplinary nature of our team, we realized that it is possible to adapt concepts of phase separation to build a dedicated organelle inside the cell that can selectively perform protein engineering by GCE. This now decouples protein engineering from canonical host function and provides a conceptually new way to study functional phase separation in biology, and push the possibilities of the field of chemical and synthetic biology. The work was accompanied by more than 20 press releases, many talk presentations and multiple awards to group members, such as the Birnstiel Award to Christopher Reinkemeier.

While our ERC COG grant focused on developing fluorescence labelling technologies, we also realized the potential of our ultrafast and ultra precise labelling technologies for other applications. With the idea to engineer antibodies with radionuclides, we were awarded an ERC proof of concept grant. This brought us into the field of antibody drug and radio conjugates, and ultimately lead to foundation of a company: Araxa Biosciences GmbH. Just recently the company fused with another Biotech startup to Veraxa Biosciences GmbH, and the company has now over 10 full time employees and keeps growing. Two former coworkers are now heads of R&D in this company, and seeing the company flourish on its way to find cures against cancer is a major success that we would have not been able to foresee when we started the ERC COG.
A scientists that looks into the interior of a cell (drawn by Nike Heinss)