Extensive de novo solid-state NMR assignments of the 33 kDa C-terminal domain of the Ure2 prion
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We present the de novo resonance assignments for the crystalline 33 kDa C-terminal domain of the Ure2 prion using an optimized set of five 3D solid-state NMR spectra. We obtained, using a single uniformly 13C, 15N labeled protein sample, sequential chemical-shift information for 74% of the N, Cα, Cβ triples, and for 80% of further side-chain resonances for these spin systems. We describe the procedures and protocols devised, and discuss possibilities and limitations of the assignment of this largest protein assigned today by solid-state NMR, and for which no solution-state NMR shifts were available. A comparison of the NMR chemical shifts with crystallographic data reveals that regions with high crystallographic B-factors are particularly difficult to assign. While the secondary structure elements derived from the chemical shift data correspond mainly to those present in the X-ray crystal structure, we detect an additional helical element and structural variability in the protein crystal, most probably originating from the different molecules in the asymmetric unit, with the observation of doubled resonances in several parts, including entire stretches, of the protein. Our results provide the point of departure towards an atomic-resolution structural analysis of the C-terminal Ure2p domain in the context of the full-length prion fibrils.
KeywordsPrion Solid-state NMR Fibrils Sequential assignment Conformational heterogeneity
This work was supported by the Agence Nationale de la Recherche (ANR-07-PCVI-0013-03, ANR-06-BLAN-0266, ANR-PCV08 321323, and ANR08-PCVI-0022-02), the ETH Zurich, the Swiss National Science Foundation (Grant 200020_124611) and the Centre National de la Recherche Scientifique. We also acknowledge support from the European Commission under the Seventh Framework Programme (FP7), contract Bio-NMR 261863.
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