Solid-State NMR Study of Amyloid Nanocrystals and Fibrils Formed by the Peptide GNNQQNY from Yeast Prion Protein Sup35p Patrick C. A. van der Wel, Jo ´ zef R. Lewandowski, and Robert G. Griffin* Contribution from the Francis Bitter Magnet Laboratory and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received December 1, 2006; E-mail: rgg@mit.edu Abstract: Sup35p is a prion protein found in yeast that contains a prion-forming domain characterized by a repetitive sequence rich in Gln, Asn, Tyr, and Gly amino acid residues. The peptide GNNQQNY7-13 is one of the shortest segments of this domain found to form amyloid fibrils, in a fashion similar to the protein itself. Upon dissolution in water, GNNQQNY displays a concentration-dependent polymorphism, forming monoclinic and orthorhombic crystals at low concentrations and amyloid fibrils at higher concentrations. We prepared nanocrystals of both space groups as well as fibril samples that reproducibly contain three (coexisting) structural forms and examined the specimens with magic angle spinning (MAS) solid-state nuclear magnetic resonance. 13 C and 15 N MAS spectra of both nanocrystals and fibrils reveal narrow resonances indicative of a high level of microscopic sample homogeneity that permitted resonance assignments of all five species. We observed variations in chemical shift among the three dominant forms of the fibrils which were indicated by the presence of three distinct, self-consistent sets of correlated NMR signals. Similarly, the monoclinic and orthorhombic crystals exhibit chemical shifts that differ from one another and from the fibrils. Collectively, the chemical shift data suggest that the peptide assumes five conformations in the crystals and fibrils that differ from one another in subtle but distinct ways. This includes variations in the mobility of the aromatic Tyr ring. The data also suggest that various structures assumed by the peptide may be correlated to the “steric zipper” observed in the monoclinic crystals. Introduction Amyloidosis, or the class of disorders associated with amyloid formation, continues to attract the attention of researchers from a wide variety of scientific disciplines. These studies are of great medical importance as a key to understanding human diseases 1 and are also of considerable scientific interest, potentially providing broader insights into fundamental biological processes such as protein (mis)folding. Unfortunately, the molecular structures of the proteinaceous aggregates, from which the name of the group is derived, remain elusive despite substantial ongoing efforts. The essential biophysical characteristics of amyloid fibrils complicate their structural characterization via conventional methods of structural biology, X-ray crystal- lography, and solution NMR, since these techniques require either crystallization or dissolution of the material of interest. For this reason, various complementary techniques are being applied to obtain insights into the mechanism behind the formation and structure of fibrillar aggregates. 2,3 These studies often involve truncated fragments of amyloidogenic proteins and are aimed at developing convenient model systems that yield detailed structural data. One such system is the yeast protein Sup35p which normally functions as a translation termination factor. 4-6 However, an aggregated form has been found to be the causative agent in the transmission of a phenotype [PSI(+)]. 7-9 The protein is the factor leading to an inheritable phenotype that could also be induced by the introduction of preformed aggregates. 8 This is analogous to the role of the prion component in human prion disorders, a class of diseases characterized by the self-propagation of pathogenic amyloid aggregates. 10 The protein’s prion-forming domain is characterized by a high percentage of glutamine and asparagine residues, similar to a class of amyloid-forming proteins implicated in various human diseases including Huntington’s disease and spinal and bulbar muscular atrophy. 11 Due to the relative convenience of the yeast organism, the Sup35p protein provides an easily accessible model system for the study of this class of disorders specifically and amyloid-forming proteins in general. 12 (1) Westermark, P. 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Published on Web 03/31/2007 10.1021/ja068633m CCC: $37.00 © 2007 American Chemical Society J. AM. CHEM. SOC. 2007, 129, 5117-5130 9 5117