In working package 1 (WP1), a site-specific labeling scheme was developed utilizing genetic code expansion facilitated by synthetic organelles for NUP98 in mammalian cells. A custom-designed FLIM setup was employed to investigate the conformations of the labeled proteins within live cells. By measuring the distance distribution of the disordered chain segments of NUP98, we demonstrated that the FG domain adopts a more expanded conformation inside the nuclear pore complex (NPC) during its functional state. This was probably the first time the dimensions of an IDP were probed inside the cell, but for sure, the first time inside functional nuclear pore complexes. The results on NUP98 published in Nature (Yu et al., 2023) have received wide international attention. The pipeline developed for NUP98 can also be used to study other IDPs in the cell.
In WP2, an in vitro reconstitute assay was performed to understand the conformational dynamics of NUP98 during phase separation. The formed NUP98 condensates undergo a liquid-to-gel transition after about five minutes. The liquid-like condensates during the initial phase transition could mimic the physiological nuclear transport pathway seen in intact NPCs within cells. The conformational dynamics of the FG domain were monitored using fluorescence anisotropy, revealing the slowing of segmental rotation as the molecular aging process continued. This outcome correlated with macroscopic observations of the condensates transitioning from a liquid-like to a gel-like state.
In WP3, the focus was on the dynamics of NUP98 in live cells utilizing high-resolution fluorescence tools. A pipeline was developed to examine the dynamics of IDPs within the cell using fluorescence anisotropy.
The results found in this project provide novel insights into the behavior and conformational dynamics of intrinsically disordered proteins both in vitro and within live cells. These findings pave the way for a deeper understanding of biological processes. In particular, the results on NUP98 have been presented at several conferences, published open access in peer-reviewed journals, and shared on various social media channels. The knowledge gained from this project has the potential to revolutionize structural biology, phase separation studies, and our comprehension of IDPs' roles in cellular processes and diseases, ultimately benefiting society through the advancement of medical research and treatments.
