title: Single molecule study of the intrinsically disordered FG-repeat nucleoporin 153.
abstract: Nucleoporins (Nups), which are intrinsically disordered, form a selectivity filter inside the nuclear pore complex, taking a central role in the vital nucleocytoplasmic transport mechanism. These Nups display a complex and nonrandom amino-acid architecture of phenylalanine glycine (FG)-repeat clusters and intra-FG linkers. How such heterogeneous sequence composition relates to function and could give rise to a transport mechanism is still unclear. Here we describe a combined chemical biology and single-molecule fluorescence approach to study the large human Nup153 FG-domain. In order to obtain insights into the properties of this domain beyond the average behavior, we probed the end-to-end distance (R(E)) of several ∼50-residues long FG-repeat clusters in the context of the whole protein domain. Despite the sequence heterogeneity of these FG-clusters, we detected a reoccurring and consistent compaction from a relaxed coil behavior under denaturing conditions (R(E)/R(E,RC) = 0.99 ± 0.15 with R(E,RC) corresponding to ideal relaxed coil behavior) to a collapsed state under native conditions (R(E)/R(E,RC) = 0.79 ± 0.09). We then analyzed the properties of this protein on the supramolecular level, and determined that this human FG-domain was in fact able to form a hydrogel with physiological permeability barrier properties.
authors.affiliation: European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany.
authors.forename: Sigrid
authors.initials: S
authors.lastname: Milles
authors.forename: Edward A
authors.initials: EA
authors.lastname: Lemke
mesh_terms: D021581:Active Transport, Cell Nucleus; D002467:Cell Nucleus / Q000378:metabolism; D006801:Humans; D028861:Nuclear Pore Complex Proteins / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D016062:Porosity; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D020449:Repetitive Sequences, Amino Acid; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: C079377:NUP153 protein, human; D028861:Nuclear Pore Complex Proteins; D010446:Peptide Fragments