The mechanisms by which intrinsically disordered proteins (IDPs) engage in rapid and highly selective binding is a subject of considerable interest and represents a central paradigm to nuclear pore complex (NPC) function. Nuclear transport receptors (NTRs) can move through the central channel of the NPC which is filled with hundreds of phenylalanine-glycine-rich nucleoporins (FG-Nups) reaching millimolar concentrations with elusive conformational plasticity. Since site-specific labeling of proteins with small but highly photostable fluorescent dyes inside cells remains the major bottleneck for directly studying protein dynamics in the cellular interior, we have now developed a semi-synthetic strategy based on novel artificial amino acids that are easily and site-specifically introduced into any protein by the natural machinery of the living cell via a newly developed thin-film synthetic organelle that equips the living cell with up to three genetic codes. This allowed us to develop an experimental approach combining site-specific fluorescent labeling of IDPs in non-fixed cells with fluorescent lifetime imaging microscopy (FLIM) to directly decipher the plasticity of FG-Nups via FRET. Our study enabled a conformational look on the condensated IDPs in the sub-resolution (roughly (50 nm)3 small cavity) cavity of the NPC. By measuring the end-to-end distances of different segments of the labeled FG-Nups using FLIM-FRET, we can extract the scaling exponent, which directly describes the conformations of FG-Nups at their functional status as well as the solvent quality in the cellular and even inner NPC environment.
