Polymorphism in an amyloid-like fibril-forming model peptide.

The conversion of peptides or proteins from their soluble forms into amyloid fibrils is frequently associated with pathological conditions ranging from neurodegenerative disorders to systemic amyloidoses. Although amyloid fibrils and non-disease-associated amyloid-like fibrils can be formed by peptides and proteins that share no sequence identity, they display several common properties. One hallmark of amyloid and amyloid-like fibrils is their highly ordered organization into a laminated cross-b structure, in which the b strands run perpendicular to the long fibril axis. Another characteristic is that the same protein or peptide can form fibrils of different morphologies. It has been suggested that the structural and morphological variability of fibrils is likely to form the molecular basis for the phenomenon of strains, and may play a role in amyloid diseases. Although the basis of amyloid fibril polymorphism is not well understood, there is spectroscopic evidence that it is accompanied by specific changes in the conformation and packing of the individual polypeptide chains. It has been shown that fibril polymorphism can partially be controlled by variation of the growth conditions and that seeds from fibrils with a particular morphology can induce the sample to polymerize into fibrils of the same morphology. Elucidation of the factors that control the polymorphism of amyloid fibrils is therefore of major importance for understanding amyloid and prion diseases at the molecular level. Herein we address the molecular basis of polymorphism using the example of the de novo designed peptide ccb-p as a model system. Previous studies have shown that ccb-p (Ac-SIRELEARIRELELRIG-NH2) adopts a three-stranded a-helical coiled-coil structure in aqueous solution at low temperatures. However, the peptide forms amyloid-like fibrils spontaneously and irreversibly upon raising the temperature. When formed from a solution buffered at pH 7.3, the b strands within the fibrils were shown to assume a laminated cross-b conformation in which the extended b strands form antiparallel b sheets. The b strands were found to be shifted by three amino acid residues from an in-register arrangement (see Figure 1b,d). We denote this arrangement as “+ 3 out-of-register” (+ 3-or). It was suggested that, in addition to the clustering of hydrophobic residues, extensive salt-bridge formation between the charged side chains of Glu and Arg is a stabilizing factor for this arrangement. Therefore, the protonation of the Glu side chains at low pH was suspected to potentially change the register. As a result of its sensitivity to the inverse third power of the internuclear distance, solid-state NMR spectroscopy, and more specifically rotational echo double-resonance (REDOR) experiments, are a powerful tool to unambiguously determine the register of constituent b strands within an amyloid fibril. The distance between the carbonyl carbon atom and the amide nitrogen atom is close to 4.2 ? if two amino acid residues are hydrogen-bonded partners, and larger than 5.5 ? otherwise. If the samples investigated are selectively labeled with a single C and a single N atom and the distance measured is about 4.2 ?, the corresponding register is unambiguously established. To investigate the structure of ccb-p amyloid-like fibrils at the atomic level, differently labeled peptides were prepared. Of particular interest in the following are the results from two compounds: for compound I the N label was located on Ala7, and for compound II on Ile2. Both samples contained, in addition, a C label on the carbonyl of Leu14. Compound I will lead to a strong REDOR effect for a + 3-or antiparallel bsheet structure, known to form at pH 7.3, and sample II for a 2-or arrangement (see Figure 1), which will be shown to form at low pH. Figure 2 shows the REDOR dephasing on fibrils of compound I prepared from solution at different pH values. The dephasing increases with increasing pH in the range from 2.0 to 7.3, indicating an increase of the abundance of the + 3or fibril polymorph, which indeed is the dominant structure at neutral pH. Figure 3 shows the REDOR data obtained from samples of compound II. For samples prepared at low pH values, a strong REDOR effect is visible, attesting the existence of a 2-or structure. The solid lines in Figures 2 and 3 indicate the best fit of the data by a model in which the dephasing is described by a superposition of the + 3-or and the 2-or register dephasing curves. This approach is justified because compound I will [*] Dr. R. Verel, I. T. Tomka, C. Bertozzi, R. Cadalbert, Prof. B. H. Meier Physical Chemistry ETH Zurich, Wolfgang-Pauli-Strasse 10, 8093, Zurich (Switzerland) Fax: (+41)44-632-1621 E-mail: beme@nmr.phys.chem.ethz.ch Homepage: http://www.ssnmr.ethz.ch