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

Abstract

The central channels of nuclear pore complexes (NPCs) in eukaryotic cells are filled with protein chains whose sequences contain characteristic phenylalanine-glycine motifs. Properties of these nanopore-confined FG-repeat sequences are of central importance to NPC function. Here we demonstrate an approach to nuclear magnetic resonance (NMR) studies of FG-repeat sequences (or other polypeptides) that are tethered within pores with diameters similar to those of NPC channels. By attaching alkyl phosphonate groups to the N-terminus of a 30-residue peptide that contains four FG repeats, called FG30, we tether FG30 chains to walls of 20-nm-diameter pores in anodic aluminum oxide (AAO) wafers through phosphonate-surface bonds. Quantitative 13C and 31P NMR measurements indicate 90 mM peptide concentrations (300 mg/ml) within the pores. NMR spectra and spin relaxation measurements show that FG30 chains are dynamically disordered and random-coil-like in buffer-filled pores over a broad temperature range. In contrast, FG30 aggregates in free solution 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 studies of polypeptides in nanopore-confined environments and show that tethering to nanopore walls can dramatically alter the self-assembly properties of an FG-repeat sequence. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/675621v2_ufig1.gif" ALT="Figure 1"> View larger version (92K): org.highwire.dtl.DTLVardef@7e0287org.highwire.dtl.DTLVardef@d81553org.highwire.dtl.DTLVardef@601639org.highwire.dtl.DTLVardef@52fc7a_HPS_FORMAT_FIGEXP M_FIG C_FIG