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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 [beta]-strands oriented orthogonal to the fibril axis ('cross [beta]' structure). We previously reported the design and characterization of a combinatorial library of de novo [beta]-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 [beta]-strands separated by reverse turns. Each [beta]-strand was designed to be seven residues long, with polar (o) and nonpolar (*) amino acids arranged with an alternating periodicity (o*o*o*o*). The initial design specified the identical polar/nonpolar pattern for all of the [beta]-strands; no strand was explicitly designated to form the edges of the resulting [beta]-sheets. With all [beta]-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 [beta]-sheet proteins, we have now redesigned the first and/or last [beta]-strands of several sequences from the original library. In the redesigned [beta]-strands, the binary pattern is changed from o*o*o*o to o*o*oKo*o (K denotes lysine). The presence of a lysine on the nonpolar face of a [beta]-strand should disfavor fibrillar structures because such structures would bury an uncompensated charge. The nonpolar [right arrow] lysine mutations, therefore, would be expected to favor monomeric structures in which the o*oKo*o 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 [beta]-strand or the C-terminal [beta]-strand (or both) is changed to lysine. Characterization of the redesigned proteins shows that they form monomeric [beta]-sheet proteins.

A variety of naturally occurring biomaterials owe their unusual structural and mechanical properties to layers of [Beta]-sheet proteins laminated between layers of inorganic mineral. To explore the possibility of fabricating novel two-dimensional protein layers, we studied the self-assembly properties of de novo proteins from a designed combinatorial library. Each protein in the library has a distinct 63 amino acid sequence, yet they all share an identical binary pattern of polar and nonpolar residues, which was designed to favor the formation of six-stranded amphiphilic [Beta]-sheets. Characterization of proteins isolated from the library demonstrates that (iii) they self assemble into monolayers at an air/water interface; (ii) the monolayers are dominated by [Beta]-sheet secondary structure, as shown by both circular dichroism and infrared spectroscopies; and (iii) the measured areas (500- 600 [[Angstrom].sup.2]) of individual protein molecules in the monolayers match those expected for proteins folded into amphiphilic [Beta]-sheets. The finding that similar structures are formed by distinctly different protein sequences suggests that assembly into [Beta]-sheet monolayers can be encoded by binary patterning of polar and nonpolar amino acids. Moreover, because the designed binary pattern is compatible with a wide variety of different sequences, it may be possible to fabricate [Beta]-sheet monolayers by using combinations of side chains that are explicitly designed to favor particular applications of novel biomaterials.

Amyloid deposits are associated with several neurodegenerative diseases, including Alzheimer's disease and the prion diseases. The amyioid 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 amyioid, 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 [Beta]-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.