Amyloids DOI: 10.1002/anie.200704896 Infectious and Noninfectious Amyloids of the HET-s(218–289) Prion Have Different NMR Spectra** Christian Wasmer, Alice Soragni, Raimon SabatØ, Adam Lange, Roland Riek, and Beat H. Meier* The prion-forming domain comprising residues 218–289 of the fungal prion HET-s has recently been shown to form amyloid fibrils at low pH in vitro which have little or no infectivity. [1] Because a molecular model for the structure of the infectious pH 7 form exists, [2] the study of the non- infectious low-pH fibrils opens an exciting possibility to address, on a molecular level, the differences that distinguish infectious from noninfectious polymorphs of the same protein. Amyloids, in general, and prions, in particular, are known to exist in different polymorphic forms, the formation of which can be controlled in vitro in part by adjusting the pH or stirring the solution. [3,4] Polymorphs can also be inheritable, a phenomenon that is intimately linked to the existence of different strains in prion diseases. Prion strains showing significantly differing biological activity have been described in yeast, [5–7] but for the HET-s prion protein of the filamentous fungus Podospora anserina no indications for polymorphism at physiological pH have been found. [1] This finding is reflected in the solid-state NMR spectra of the prion-forming C-terminal domain of HET-s, the fragment HET-s(218–289), for which narrow NMR linewidths for both 13 C and 15 N resonances have been found, and no indications for peak doubling were detected. [8] Furthermore, no variation of chemical shifts has been found for samples from several different preparations. For the pH 3 fibrils, whose NMR spectra are described in this communication, there is, how- ever, evidence from electron microscopy that several poly- morphs indeed coexist, all of which are different from the pH 7 form. [1] The C-terminal fragment comprising residues 218 to 289 forms the proteinaseK-resistant part of the fibrils [9] and has the sequence KIDAIVGRNSAKDIRTEERARVQLGN- VVTAAALHGGIRISDQTTNSVETVVGKGESRVLIGN- EYGGKGFWDN. This fragment is necessary and sufficient for prion infectivity [10] and forms infectious amyloid fibrils at pH 7 in vitro. [9] The well-resolved NMR spectra of the HET- s(218–289) pH 7 fibrils allowed for an almost complete sequence-specific assignment of the NMR resonances for the rigid parts. [11] The chemical shift information together with additional biophysical data have been used to propose a structural model with four b strands, b 1 –b 4 . These four strands form a b-solenoid fold with two repeating strand–turn–strand motifs (b 1 –b 2 and b 3 –b 4 ) forming two turns of the solenoid. This model is supported by recent electron microscopy data which show a mass-per-length ratio consistent with two layers of b strands per HET-s(218–289) subunit. [12] M-HET-s(218–289)-H 6 was recombinantly expressed and purified according to a previously described procedure, [9,11] and fibrillization was carried out at pH 3, as described in detail in the Supporting Information. The pH 3 fibrils were found to be more stable than fibrils formed at pH 2, the pH at which most of the experiments in reference [1] were per- formed. However, the pH 2 and pH 3 fibrils behave very similarly (they have almost identical aggregation kinetics, both induce thioflavin T fluorescence, and they show the same morphology in electron micrographs), and it was confirmed that the pH 3 form is not infectious. [22] The 13 C– 13 C proton-driven spin diffusion (PDSD) spec- trum of the pH 3 fibrils is shown in Figure 1 along with the corresponding spectrum of pH 7 fibrils. After their formation, the pH 3 fibrils were washed in pure water. While fibrilliza- tion at neutral pH yields the pH 7 conformational state, [1] the pH 3 form is stable at higher pH and no pH 7 fibrils could be detected in our experiments. The spectra of the pH 3 fibrils are clearly different from those of the pH 7 form, indicating a different molecular structure; the spectral resolution is some- what lower, indicating higher disorder: The pH 3 fibrils exhibit typical linewidths between 128 Hz and 202 Hz com- pared to linewidths of less than 100 Hz for the pH 7 fibrils (only well-resolved peaks were analyzed). The reduced resolution made the sequential resonance assignment diffi- cult. Nevertheless, we have been able to identify and tentatively assign 22 spin systems, each corresponding to an amino acid residue, by through-bond 13 C– 13 C TOBSY spec- troscopy [13,14] (Figure 2), in combination with the PDSD and HETCOR spectra (Figure 1). The 22 spin systems detected in the rigid parts of the fibril consist of 3 A, 1 D (or N), 2 E, 2 G, 1 H, 2 I, 1 K, 1 L, 1 R, 2 S, 2 T, and 4 V. The complete TOBSY spectrum and the assigned chemical shifts are given in the Supporting Information. For 16 of these spin systems, both the C a and C b chemical shifts were assigned and the differences in their secondary chemical shifts, d C aÀd C b, are shown in Figure 3. For all of the spin systems negative values were [*] C. Wasmer, A. Soragni, Dr. A. Lange, Prof. R. Riek, Prof. B. H. Meier Laboratorium für Physikalische Chemie ETH Zurich, 8093 Zurich (Switzerland) Fax: (+ 41)446-321-621 E-mail: beme@ethz.ch Dr. R. SabatØ Laboratoire de Genetique Moleculaire des Champignons IBGC UMR CNRS 5095, UniversitØ de Bordeaux 2 Bordeaux (France) [**] We thank S. J. Saupe, H. Van Melckebeke, A. Siemer, and M. Ernst for helpful discussions. This research was supported by the Swiss National Science Foundation and the ETH Zurich through the TH grant system. Supporting information for this article is available on the WWW under http://www.angewandte.org or from the author. Angewandte Chemie 5839 Angew. Chem. Int. Ed. 2008, 47, 5839–5841 # 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim