Wednesday, June 04, 2008

J. Am. Chem. Soc., 130 (23), 7172–7173, 2008.

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

Mirko Sackewitz,† Holger A. Scheidt,† Grit Lodderstedt,† Angelika Schierhorn,‡ Elisabeth Schwarz,† and Daniel Huster*†§

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 β-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−13Cα 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.

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