Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - SMPFv2.0 (Next generation single molecule protein fluorescence)

Teaser

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...

Summary

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. This is in particular important when studying complex biological processes, such as those of very dynamics building blocks. Noteably, in more complex organisms like humans, very dynamic proteins, so called intrinsically disordered proteins (IDPs) are enriched, likely because multiple tasks can be easier encoded into those vs more static or solid building blocks.
Despite all progress, single molecule fluorescence science suffers from the need for site-specific labelling and the intrinsic low throughput. In this project, we aim to improve single molecule science, by integrating chemical biology and microfluidic tools into a synergistic process on how to study biological building blocks with high resolution at the single molecule level. The tools will be directly used to study a set of IDPs highly relevant to a fundamental function of the eukaryotic cell, which in turn will also feedback on the tool development.

Work performed

We have developed a new method to perform superresolution microscopy with residue-specific resolution. The work was published in Angewandte (2016), was selected as a hot paper and featured as a cover article. It is based on an improvement of Genetic code expansion technology (GCE), which permits to site-specifically install Click reactive noncanonical amino acids (ncAAs). Those were subsequently labelled with a tetrazine containing sDNA strand (template strand). Following a protocol of the previously described DNA-PAINT technology (Jungman et al, Nature Methods 2014), another complementary sDNA strand containing a dye can transiently anneal to the template strand, which then enables generation of a super-resolution image by means of localization microscopy techniques. We have now also been able to decipher the relevance of nucleoporin glycosylation for differential binding mechanisms to nuclear transport receptors.
Until now three reviews related to this ERC project have been published to further disseminate the work.

Final results

Site-specifically encoding ncAAs by means of GCE has become one of the most versatile tools in protein engineering. In particular, the use of the Amber suppressor tRNA/tRNA synthetase (tRNA, RS) pair from Methanosarcina has greatly contributed to this success. We discovered that the widely used RS from this species contains a nuclear localization signal. While this has no apparent impact when GCE is performed in prokaryotes, in eukaryotes this can lead to accumulation of the tRNA/RS in the nucleus. There it cannot easily participate in translation, which can lower efficiency and increase background in fluorescence labelling studies. The finding was published in Angewandte 2016 and a patent application has been filed.