Deciphering the mechanism(s) of -sheet mediated self-assembly is essential for understanding amyloid fibril formation and for the fabrication of polypeptide materials. Herein, we report a simple peptidomimetic that self-assembles into polymorphic -sheet quaternary structures including protofilaments, filaments, fibrils, and ribbons that are reminiscent of the highly ordered structures displayed by the amyloidogenic peptides A , calcitonin, and amylin. The distribution of quaternary structures can be controlled by and in some cases specified by manipulating the pH, buffer composition, and the ionic strength. The ability to control -sheet-mediated assembly takes advantage of quaternary structure dependent pK a perturbations. Biophysical methods including analytical ultracentrifugation studies as well as far-UV circular dichroism and FT-IR spectroscopy demonstrate that linked secondary and quaternary structural changes mediate peptidomimetic self-assembly. Electron and atomic force microscopy reveal that peptidomimetic assembly involves numerous quaternary structural intermediates that appear to self-assemble in a convergent fashion affording quaternary structures of increasing complexity. The ability to control the assembly pathway(s) and the final quaternary structure(s) afforded should prove to be particularly useful in deciphering the quaternary structural requirements for amyloid fibril formation and for the construction of noncovalent macromolecular structures.The self-assembly of peptides and proteins into noncovalent -sheet rich quaternary structures, including fibrils, has attracted the attention of numerous laboratories owing to their association with neurodegenerative disease and their interesting structures. [1][2][3][4][5][6][7][8][9][10] In both amyloid and prion diseases a normally soluble protein or proteolytic fragment undergoes a conformational change either prior to, or coincident with, its self-assembly into -sheet rich fibrils, implicated as the causative agent in numerous neurodegenerative diseases by genetic linkage. [11][12][13][14][15][16][17][18][19] Previous studies on amyloid fibril assembly establish the presence of quaternary structural intermediates, which appear to undergo convergent assembly into intermediates of increasing complexity until amyloid fibrils are ultimately afforded. [20][21][22][23][24][25][26][27][28] The differing quaternary structures observed could explain the strains char- ‡ Laboratory of Molecular Biology. (3) Yamada, N.; Katsuhiko, A.; Naito, M.; Matsubara, K.; Koyama, E. J. Am. Chem. Soc. 1998, 120, 12192-12199. (4) Janek, K.; Behlke, J.; Zipper, J.; Fabian, H.; Georgalis, Y.; Beyemmann, M.; Bienert, M.; Krause, E.

The L55P transthyretin (TTR) familial amyloid polyneuropathy-associated variant is distinct from the other TTR variants studied to date and the wild-type protein in that the L55P tetramer can dissociate to the monomeric amyloidogenic intermediate and form fibril precursors under physiological conditions (pH 7.0, 37°C). The activation barrier associated with L55P-TTR tetramer dissociation is lower than the barrier for wild-type transthyretin dissociation, which does not form fibrils under physiological conditions. The L55P-TTR tetramer is also very sensitive to acidic conditions, readily dissociating to form the monomeric amyloidogenic intermediate between pH 5.5-5.0 where the wildtype TTR adopts a nonamyloidogenic tetrameric structure. The formation of the L55P monomeric amyloidogenic intermediate involves subtle tertiary structural changes within the -sheet rich subunit as discerned from Trp fluorescence, circular dichroism analysis, and ANS binding studies. The assembly of the L55P-TTR amyloidogenic intermediate at physiological pH (pH 7.5) affords protofilaments that elongate with time. TEM studies suggest that the entropic barrier associated with filament assembly (amyloid fibril formation) is high in vitro, amyloid being defined by the laterally assembled four filament structure observed by Blake upon isolation of "fibrils" from the eye of a FAP patient. The L55P-TTR protofilaments formed in vitro bind Congo red and thioflavin T (albeit more weakly than the fibrils produced at acidic pH), suggesting that the structure observed probably represents an amyloid precursor. The structural continuum from misfolded monomer through protofilaments, filaments, and ultimately fibrils must be considered as a possible source of pathology associated with these diseases.There are several human neurodegenerative diseases putatively caused by extracellular protein deposition (1). The most familiar of these include the amyloid diseases, e.g. Alzheimer's disease and senile systemic amyloidosis (2-7). In all amyloid diseases, proteins undergo conformational changes either before or coincident with their self-assembly into highly ordered amyloid fibrils. Amyloid fibrils having a cross -sheet quaternary structure are typically deposited in the brain and/or in peripheral tissues and have been linked to neurodegeneration and/or organ dysfunction by genetic evidence (2,(8)(9)(10)(11). The amyloid hypothesis implicates amyloid fibrils as the causative agent in amyloid diseases through a gain of function phenotype. (1,7,9,(12)(13)(14). Recent results from several laboratories, including our own, demonstrate the existence of preamyloid quaternary structural intermediates (protofilaments), implying that the whole process of amyloid fibril formation must be considered as a possible cause of pathology, including the first step in the process, which is protein misfolding in the case of structurally well-defined amyloid proteins (14-21).Understanding the mechanism of how a folded protein is converted into an insoluble amyloid fibr...