De novo beta-sheet proteins: Amyloid-like fibrils, self-assembling monolayers and monomeric structures

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 structural determinants in amyloid fibril formation, this thesis first describes the characterization of a combinatorial library of de novo designed sequences. 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 (chapter 2).; A variety of naturally occurring biomaterials owe their unusual structural and mechanical properties to layers of β-sheet proteins laminated between layers of inorganic mineral. To explore the possibility of fabricating novel two-dimensional protein layers, the self-assembly properties of the novel β-sheet proteins were studied. Characterization of proteins isolated from the library demonstrates that (i) they self assemble into monolayers at an air/water interface; (ii) the monolayers are dominated by β-sheet secondary structure; and (iii) the measured areas (500–600 Å 2) of individual protein molecules in the monolayers match those expected for proteins folded into amphiphilic β-sheets. The finding that similar structures are formed by distinctly different protein sequences suggests that assembly into β-sheet monolayers can be encoded by binary patterning of polar and nonpolar amino acids (chapter 3).; Protein engineering efforts to prevent the novel β-sheet proteins from self-assembling are also described. In a rational approach, negative design features were incorporated to discourage the fibril formation. The oligomerization of the β-sheet structures was disrupted when a charged group (Lysine) was placed at selected positions (chapter 4). In a combinatorial approach, a fluorescence screen based on the green fluorescence protein (GFP) was developed. In this screen, GFP fusion partners fluorescence only when the fusion partner is soluble and monomeric. This screen was utilized to select monomeric mutants of the original amyloid-like sequences from pools of mutants generated by random mutagenesis (chapter 5).