Bilayer Fibril Formation by Genetically Engineered Polypeptides:  Preparation and Characterization

Abstract

A de novo, genetically engineered 687 residue polypeptide expressed in E. coli has been found to form highly rectilinear, beta-sheet containing fibrillar structures. Tapping-mode atomic force microscopy, deep-UV Raman spectroscopy, and transmission electron microscopy definitively established the tendency of the fibrils to predominantly display an apparently planar bilayer or ribbon assemblage. The ordered self-assembly of designed, extremely repetitive, high molecular weight peptides is a harbinger of the utility of similar materials in nanoscience and engineering applications.

β‐sheet folding of 11‐kDa fibrillogenic polypeptide is completely aggregation driven

“…22,23 After the final purification step, dialysis against doubly distilled water, the YE8 solution was centrifuged at 15000g for 45 min to remove polypeptide aggregates. The supernatant was diluted with either distilled water (pH 6.5) or 10-mM sodium formate buffer (pH 3.5) to yield initial solutions with a concentration of 44 lM.…”

“…We have demonstrated 22,23 that, after lysine carbamylation, a de novo, genetically engineered 687-residue peptide comprised of 21 repeats of a 32 amino acid monomer unit GH 6 [(GA) 3 GY(GA) 3 GE(GA) 3 GH(GA) 3 GK] 21 GAH 6 (YEHK21) self-assembles into a well-defined antiparallel b-sheet structure stabilized by intramolecular hydrogen bonds. YEHK21 forms a gelatinous phase in solution and fibril-type ribbons on welldefined, hydrophobic surfaces.…”

“…YEHK21 forms a gelatinous phase in solution and fibril-type ribbons on welldefined, hydrophobic surfaces. 22 Here we report on the design, synthesis and folding of a congener consisting of 8 repeats of the 16 amino acid monomer, YE8, GH 6 [(GA) 3 GY(GA) 3 GE] 8 GAH 6 where both the N-and C-terminii bear hexahistidinyl tracts. It was hypothesized that the GAGAGA repeats form the 16 antiparallel b-strands of the b-sheet structure with differentiated turn groups along the edges ( Figure 1): one edge with arrays of tyrosine residues (Y) and the other with charged glutamic acids (E).…”

“…19 Recently, subsequent to reports of a number of short peptides (9-16 residues in length) that fold into two-stranded antiparallel b-sheets (''b hairpins'') or three-stranded antiparallel bsheets, 20,21 we developed methods for the construction of much larger b-sheet forming peptides. 22,23 Genetic engineering affords excellent opportunities to verify the mechanism of folding and fibrillation by tuning selectively the primary polypeptide sequence and by testing the influence of those changes on kinetic and equilibrium properties. Criteria to select appropriate turn and strand residues and to avoid aggregation are available.…”

Evaluation of Conformation and Association Behavior of Multivalent Alanine-Rich Polypeptides

“…Accordingly, the versatility of these synthetic methods has permitted the production of a variety of protein polymers with well-defined secondary structures designed for a specific function or application. Numerous studies have focused on the synthesis of repetitive amino acid sequences that adopt β-sheet, coiledcoil or random coil structures (17,19,20,(22)(23)(24)(25)(26)(27)(28)(29), although there have been fewer reports about sequences designed to adopt monomeric α-helical secondary structures (15,16,18,30).…”