Structural and Dynamical Characterization of Fibrils from a Disease-Associated Alanine Expansion Domain Using Proteolysis and Solid-State NMR Spectroscopy

Abstract

The nuclear poly(A) binding protein PABPN1 possesses a natural 10 alanine stretch that can be extended to 17 Ala by codon expansion. The expansions are associated with the disease oculopharyngeal muscular dystrophy (OPMD), which is characterized histopathologically by intranuclear fibrillar deposits. Here, we have studied the Ala extended fibrillar N-terminal fragment of PABPN1, (N-(+7)Ala), comprising 152 amino acids. At natural abundance, cross-polarized 13C MAS NMR spectra are dominated by the three Ala signals with characteristic beta-sheet chemical shifts. In contrast, directly polarized 13C MAS spectra show a multitude of narrow lines, suggesting a large portion of highly mobile sites. Proteolytic cleavage of the protein combined with MALDI-TOF mass spectrometry revealed a protease-resistant peptide encompassing residues 13/14 to 50-52 with the poly-Ala stretch in the center. Measurements of the 1H-13Calpha dipolar couplings of 13C/15N-labeled N-(+7)Ala revealed high order parameters of 0.77 for the poly-Ala stretch of the fibril, while the majority of the residues of N-(+7)Ala exhibited very low order parameters between 0.06 and 0.15. Only some Gly residues that are flanking the Ala-rich region had significant order parameters of 0.47. Thus, site-specific dynamic mapping represents a useful tool to identify the topology of fibrillar proteins.

Dynamics of Amyloid β Fibrils Revealed by Solid-state NMR

“…This is also consistent with previous evidence that a salt bridge between Asp 23 and Lys 26 (14,26) stabilizes this short connection and prevents larger backbone motions. The high order parameter values observed for the two central ␤-sheet regions also correspond well with literature data on the order of PABPN1 fibrils (19) and fibrils of the human prion protein (20).…”

“…First, the measured order parameters for the N terminus are still significantly higher than those associated with a purely thermally fluctuating random coil (which is typically ϳ0.1-0.3 even under solid-state conditions) (19). Moreover, such low order parameters would be expected to abolish or to strongly attenuate the cross-polarization signal of these protein parts (27), which was not observed here.…”

“…Interestingly, the molecular dynamics of A␤ fibrils, which is an essential part of the structural biology of amyloid structures, has not been comprehensively studied. In fact, only a very few reports on fibrillar dynamics in general exist so far (15,19,20). These studies showed that the investigation of the fibril dynamics can significantly support the structural analysis and further characterize the respective structural elements.…”

Polyalanine-independent Conformational Conversion of Nuclear Poly(A)-binding Protein 1 (PABPN1)

“…A slightly reduced affinity toward nucleic acid substrates has been observed for typical amyloid-like fibrils from fusions of the N-terminal domain of PABPN1 and the nucleic acid binding model protein CspB (39). Furthermore, a previous NMR analysis of amyloid-like fibrils formed by the N-terminal domain revealed that only a rather short segment comprising ϳ35 residues can be sufficient to make up the fibrillar core (40). In such a structure, folded domains could be preserved.…”

Festkörper‐NMR‐spektroskopische Untersuchungen an Aβ‐Protofibrillen: Nachweis einer Umgestaltung der β‐Faltblätter bei der Ausreifung von Amyloidfibrillen

“…[9] Insgesamt wurden acht Ab(1-40)-Peptide mit verschiedenen Isotopenmarkierungen chemisch synthetisiert. Dabei decken die 15 Nund 13 C-Markierungen insgesamt 30 Aminosäuren (AS) aus allen Strukturregionen des Peptids ab (Abbildung S2 in den Hintergrundinformationen). Mit diesen Peptiden wurden anschließend die jeweiligen Proben von B10AP-stabilisierten Ab(1-40)-PFs in vitro hergestellt.…”