Amyloid deposits are associated with several neurodegenerative diseases, including Alzheimer’s disease and the prion diseases. The amyloid fibrils isolated from these different diseases share similar structural features. However, the protein sequences that assemble into these fibrils differ substantially from one disease to another. To probe the relationship between amino acid sequence and the propensity to form amyloid, we studied a combinatorial library of sequences designed de novo . All sequences in the library were designed to share an identical pattern of alternating polar and nonpolar residues, but the precise identities of these side chains were not constrained and were varied combinatorially. The resulting proteins self-assemble into large oligomers visible by electron microscopy as amyloid-like fibrils. Like natural amyloid, the de novo fibrils are composed of β-sheet secondary structure and bind the diagnostic dye, Congo red. Thus, binary patterning of polar and nonpolar residues arranged in alternating periodicity can direct protein sequences to form fibrils resembling amyloid. The model amyloid fibrils assemble and disassemble reversibly, providing a tractable system for both basic studies into the mechanisms of fibril assembly and the development of molecular therapies that interfere with this assembly.

One hundred years ago, Alois Alzheimer observed a relationship between cognitive impairment and the presence of plaque in the brains of patients suffering from the disease that bears his name. The plaque was subsequently shown to be composed primarily of a 42-residue peptide called amyloid β (Aβ) 42. Despite the importance of Aβ42 aggregation in the molecular etiology of Alzheimer's disease, the amino acid sequence determinants of this process have yet to be elucidated. Although stretches of hydrophobic residues in the C-terminal half of Aβ42 have been implicated, the mechanism by which these residues promote aggregation remains unclear. In particular, it is not known whether the side chains of these hydrophobic residues mediate specific interactions that direct self-assembly or, alternatively, whether hydrophobicity per se at these positions is sufficient to promote aggregation. To distinguish between these two possibilities, we substituted 12 hydrophobic residues in the C-terminal half of Aβ42 with random nonpolar residues. The mutant sequences were screened by using a fusion of Aβ42 to GFP. Because aggregation of Aβ42 prevents folding of the GFP reporter, this screen readily distinguishes aggregating from nonaggregating variants of Aβ42. Application of the screen demonstrated that, despite the presence of 8–12 mutations, all of the sequences aggregated. To confirm these results, several of the mutant sequences were prepared as synthetic peptides and shown to form amyloid fibrils similar to those of WT Aβ42. These findings indicate that hydrophobic stretches in the sequence of Aβ42, rather than specific side chains, are sufficient to promote aggregation.

Amyloid fibrils are associated with a variety of neurodegenerative maladies including Alzheimer's disease and the prion diseases. The structures of amyloid fibrils are composed of β-strands oriented orthogonal to the fibril axis (“cross β” structure). We previously reported the design and characterization of a combinatorial library ofde novoβ-sheet proteins that self-assemble into fibrillar structures resembling amyloid. The libraries were designed by using a “binary code” strategy, in which the locations of polar and nonpolar residues are specified explicitly, but the identities of these residues are not specified and are varied combinatorially. The initial libraries were designed to encode proteins containing amphiphilic β-strands separated by reverse turns. Each β-strand was designed to be seven residues long, with polar (○) and nonpolar (●) amino acids arranged with an alternating periodicity (○●○●○●○). The initial design specified the identical polar/nonpolar pattern for all of the β-strands; no strand was explicitly designated to form the edges of the resulting β-sheets. With all β-strands preferring to occupy interior (as opposed to edge) locations, intermolecular oligomerization was favored, and the proteins assembled into amyloid-like fibrils. To assess whether explicit design of edge-favoring strands might tip the balance in favor of monomeric β-sheet proteins, we have now redesigned the first and/or last β-strands of several sequences from the original library. In the redesigned β-strands, the binary pattern is changed from ○●○●○●○ to ○●○K○●○ (K denotes lysine). The presence of a lysine on the nonpolar face of a β-strand should disfavor fibrillar structures because such structures would bury an uncompensated charge. The nonpolar → lysine mutations, therefore, would be expected to favor monomeric structures in which the ○●○K○●○ sequences form edge strands with the charged lysine side chain accessible to solvent. To test this hypothesis, we constructed several second generation sequences in which the central nonpolar residue of either the N-terminal β-strand or the C-terminal β-strand (or both) is changed to lysine. Characterization of the redesigned proteins shows that they form monomeric β-sheet proteins.

Combinatorial libraries ofde novoamino acid sequences can provide a rich source of diversity for the discovery of novel proteins. Randomly generated sequences, however, rarely fold into well ordered protein-like structures. To enhance the quality of a library, diversity must be focused into those regions of sequence space most likely to yield well folded structures. We have constructed focused libraries ofde novosequences by designing the binary pattern of polar and nonpolar amino acids to favor structures that contain abundant secondary structure, while simultaneously burying hydrophobic side chains in the protein interior and exposing hydrophilic side chains to solvent. Because binary patterning specifies only the polar/nonpolar periodicity, but not the identities of the side chains, detailed structural features, including packing interactions, cannot be designeda priori. Can binary patterned libraries nonetheless encode well folded proteins? An unambiguous answer to this question requires determination of a 3D structure. We used NMR spectroscopy to determine the structure of S-824, a novel protein from a recently constructed library of 102-residue sequences. This library is “naïve” in that it has not been subjected to high-throughput screens or directed evolution. The experimentally determined structure of S-824 is a four-helix bundle, as specified by the design. As dictated by the binary-code strategy, nonpolar side chains are buried in the protein interior, and polar side chains are exposed to solvent. The polypeptide backbone and buried side chains are well ordered, demonstrating that S-824 isnota molten globule and forms a unique structure. These results show that amino acid sequences that have neither been selected by evolution, nor designed by computer, nor isolated by high-throughput screening, can form native-like structures. These findings validate the binary-code strategy as an effective method for producing vast collections of well foldedde novoproteins.