Solid-State NMR Studies of the Fibril Forming Protein PABPN1

Abstract

An N-terminal extension of the alanine-rich stretch of the nuclear poly(A) binding protein PABPN1 leads to the disease oculopharyngeal muscular dystrophy. Histochemically, the disease is characterized by intranuclear inclusions of amyloid-like fibrils. For the study of the structure and dynamics of PABPN1 by solid-state NMR a N-(+7)Ala variant of PABPN1(1-132) was uniformly 13C and 15N labelled. Using different NMR techniques (direct, cross polarization, INEPT transfer), variations in the protein dynamics were revealed. Especially the Ala signals show a largely increased intensity in cross-polarized (CP) spectra indicating the presence of a rigid structure for the poly-alanine segment. Most amino acid types s of PABPN1(1-132) are in a random coil conformation. In contrast, for the alanine residues of the protein it was possible to distinguish between two different conformations: A β-sheet conformation was preferentially observed in CP excited spectra and a random coil structure in directly excited or INEPT spectra. Therefore, we conclude that poly-alanine segment of PABPN1 adopts a β-sheet conformation. Additionally, a magnetization transfer form Ala to Gly, which are located next to the alanine stretch, was observed in 2D proton-driven spin diffusion experiments. This indicates that the fibril is extended over the entire poly-alanine segment. To investigate the site-specific dynamics along the backbone of PABPN1(1-132) the CH order parameters for the Cα atoms were measured in DIPSHIFT experiments While the non-fibrillar part of the proteins exhibit low order parameters and has to be considered as quite flexible, the order parameter for the Ala stretch including neighbouring Gly residues are largely increased and characterize this domain as rather rigid. We conclude that the poly-alanine segment forms a β-sheet conformation and forms rigid fibrils, while the rest of PABPN1(1-132) adopts a highly mobile and flexible random coil conformation.