title: Engineering of beta-propeller protein scaffolds by multiple gene duplication and fusion of an idealized WD repeat.
abstract: The ability to design specific amino acid sequences that fold into desired structures is central to engineering novel proteins. Protein design is also a good method to assess our understanding of sequence-structure and structure-function relationships. While beta-sheet structures are important elements of protein architecture, it has traditionally been more difficult to design beta-proteins than alpha-helical proteins. Taking advantage of the tandem repeated sequences that form the structural building blocks in a group of beta-propeller proteins; we have used a consensus design approach to engineer modular and relatively large scaffolds. An idealized WD repeat was designed from a structure-based sequence alignment with a set of structural guidelines. Using a plasmid sequential ligation strategy, artificial concatemeric genes with up to 10 copies of this idealized repeat were then constructed. Corresponding proteins with 4 through to 10 WD repeats were soluble when over-expressed in Escherichia coli. Notably, they were sufficiently stable in vivo surviving attack from endogenous proteases, and maintained a homogeneous, non-aggregated form in vitro. The results show that the beta-propeller scaffold is an attractive platform for future engineering work, particularly in experiments in which directed evolution techniques might improve the stability of the molecules and/or tailor them for a specific function.
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, England, UK.
authors.forename: Maryam
authors.initials: M
authors.lastname: Nikkhah
authors.forename: Zahra
authors.initials: Z
authors.lastname: Jawad-Alami
authors.forename: Mykhaylo
authors.initials: M
authors.lastname: Demydchuk
authors.forename: Duncan
authors.initials: D
authors.lastname: Ribbons
authors.forename: Massimo
authors.initials: M
authors.lastname: Paoli
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D020440:Gene Duplication; D015202:Protein Engineering; D011506:Proteins / Q000737:chemistry / Q000235:genetics*
chemical_list: D011506:Proteins