De novo amyloid proteins from designed combinatorial libraries

Abstract

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 selfassemble into large oligomers visible by electron microscopy as amyloid-like fibrils. Like natural amyloid, the de novo fibrils are composed of ␤-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.

Cofactor binding and enzymatic activity in an unevolved superfamily of de novo designed 4‐helix bundle proteins

“…8 We have used the binary code strategy to construct several libraries of a-helical or b-sheet proteins. [8][9][10][11][12] Because the designed b-sheet proteins often form insoluble aggregates, our current work on artificial superfamilies focuses on a-helical structures. 11 The binary patterned design of a-helical sequences places a hydrophobic amino acid every three or four residues, generating the following pattern: OlOO llOOlOOllO, where O and l represent polar and nonpolar residues, respectively.…”

“…[8][9][10][11][12] Because the designed b-sheet proteins often form insoluble aggregates, our current work on artificial superfamilies focuses on a-helical structures. 11 The binary patterned design of a-helical sequences places a hydrophobic amino acid every three or four residues, generating the following pattern: OlOO llOOlOOllO, where O and l represent polar and nonpolar residues, respectively. 9,10 Consistent with the 3.6 residues/turn periodicity of a canonical a-helix, this pattern encodes a-helices that are amphiphilic.…”

Investigating the Effects of Mutations on Protein Aggregation in the Cell

“…In addition to its significance in cell biology and medicine, protein aggregation is a fundamental problem in biotechnology. For example, the heterologous expression of proteins in bacteria, the de novo design of novel proteins or the rational modification of existing proteins, is frequently frustrated by the fact that the polypeptide chains aggregate into large assemblies, including inclusion bodies or amyloid fibrils (5)(6)(7).…”

Combinatorial approaches: A new tool to search for highly structured β-hairpin peptides

“…Synthetic peptide combinatorial libraries have arisen as a source of new lead compounds (4); combinatorial libraries of genes have provided new proteins and protein domains (5,6); and peptide libraries built on ␣-helical scaffolds have appeared as a useful strategy for the identification of new antimicrobial and catalytic synthetic ␣-helical peptides (7,8).…”