Rationally designed mutations convertde novoamyloid-like fibrils into monomeric β-sheet proteins

Abstract

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 ␤-strands oriented orthogonal to the fibril axis (''cross ␤'' structure). We previously reported the design and characterization of a combinatorial library of de novo ␤-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 ␤-strands separated by reverse turns. Each ␤-strand was designed to be seven residues long, with polar (E) and nonpolar (F) amino acids arranged with an alternating periodicity (EFEFEFE). The initial design specified the identical polar͞nonpolar pattern for all of the ␤-strands; no strand was explicitly designated to form the edges of the resulting ␤-sheets. With all ␤-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 edgefavoring strands might tip the balance in favor of monomeric ␤-sheet proteins, we have now redesigned the first and͞or last ␤-strands of several sequences from the original library. In the redesigned ␤-strands, the binary pattern is changed from EFEFEFE to EFEKEFE (K denotes lysine). The presence of a lysine on the nonpolar face of a ␤-strand should disfavor fibrillar structures because such structures would bury an uncompensated charge. The nonpolar 3 lysine mutations, therefore, would be expected to favor monomeric structures in which the EFEKEFE 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 ␤-strand or the C-terminal ␤-strand (or both) is changed to lysine. Characterization of the redesigned proteins shows that they form monomeric ␤-sheet proteins.

In silico Designing of Protein Rich in Large Neutral Amino Acids Using Bovine αs1 Casein for Treatment of Phenylketonuria

“…It allows the hydrophobic amino acids to burry inside and hydrophilic amino acids to expose to the solvent [21,22]. The order of arrangement for β-sheets was with alternating Polar (P) and Non-polar (N) residues, i.e., PNPNPNP [23,24].…”

Charge substitution shows that repulsive electrostatic interactions impede the oligomerization of Alzheimer amyloid peptides

“…The first 16 residues of Ab are rich in charged residues and the rest of the sequence contains two stretches of hydrophobic residues (17)(18)(19)(20)(21) and (30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40), believed to be key for oligomerization, separated by a region containing two acidic residues (Glu 22 and Asp 23) and a basic one (Lys 28) ( Table 1). Ab oligomerization is highly pH dependent.…”

Amyloidogenic sequences in native protein structures

“…27,49 proteins as well as with mutated forms of naturally occurring proteins, have elucidated features of polypeptide sequence which inhibit aggregation and fibril formation-so-called ''negative design'' features. [52][53][54] The term ''structural gatekeeper'' was coined by Otzen et al 55 to describe charged side chains that prevent aggregation by interrupting contiguous stretches of hydrophobic residues in the primary sequence. A systematic survey of edge strands in a large sample of all-b proteins revealed several features which would prevent further b-sheet interactions via main chain hydrogen-bonding, such as bbulges, proline residues, very short edge strands, tertiary contacts with loop regions and charged residues occurring in positions unfavourable for further strand interaction.…”