Effects of Nanopore Confinement on the Conformational, Dynamical, and Self-Assembly Properties of an FG-Repeat Peptide

The central channels of nuclear pore complexes (NPCs) in eukaryotic cells contain a high concentration of protein chains with characteristic phenylalanine-glycine motifs, called FG-repeat domains, tethered to the channel walls. Passage of macromolecules and macromolecular assemblies between the cell nucleus and the cytoplasm depends on the dynamics of the FG-repeat domains and their interactions with nuclear transport receptors. Yet FG-repeat domains are highly aggregation-prone in free solution, forming amyloid-like fibrils at relatively low concentrations. Here we describe an approach to studying the properties of FG-repeat domains (or other polypeptides) when tethered to the walls of pores with diameters similar to those of NPC channels. By attaching alkyl phosphonate groups to the N-termini of 30-residue FG30 peptides that contain four FG repeats, we tether FG30 chains to walls of 20-nm-diameter pores in anodic aluminum oxide (AAO) wafers through phosphonate-surface bonds. Quantitative ¹³ C and ³¹ P nuclear magnetic resonance (NMR) measurements indicate 90 mM peptide concentrations (300 mg/ml) within the pores. One- and two-dimensional NMR spectra and nuclear spin relaxation measurements show that FG30 chains are dynamically disordered and random-coil-like in buffer-filled pores over a broad temperature range, with no sign of aggregation over many weeks despite the high intrapore concentrations. In contrast, the same FG30 peptide aggregates in free solution at 24° C at concentrations above 2 mM, forming structurally ordered fibrils according to electron microscopy and NMR measurements. These results demonstrate the utility of AAO as a scaffold for studying the properties of FG-repeat domains under conditions that mimic those within NPC channels, show that NMR measurements can elucidate these properties, and suggest that tethering to nanopore walls dramatically alters the self-assembly properties of FG-repeat domains, with potentially important implications for NPC function.