Economy in Protein Design:  Evolution of a Metal-Independent ββα Motif Based on the Zinc Finger Domains

Abstract

An iterative design process involving the synthesis and structural analyses of five polypeptides patterned after the zinc finger domains is described. This process has led to the development of a metal-independent folded ββα motif, BBA1 . In contrast to the zinc fingers and other naturally occurring peptides of similar size, this small monomeric structure folds without the assistance of metal cation ligation or disulfide bridges. To probe the effect of metal binding on the secondary and tertiary structure of peptides throughout the design process, a non-standard amino acid 3-(1,10-phenanthrol-2-yl)- l -alanine (Fen) was incorporated and its unique chromophore utilized for circular dichroism (CD) analysis. Advanced designs were analyzed by both CD and 2D NMR. The solution structure of BBA1 was determined using NOE restrained simulated annealing. The average RMSD for the backbone atoms of residues 1−22 is 0.9 ± 0.3 Å. Analysis of the resulting structure reveals that the α-helix and β-hairpin are associated via a well-defined hydrophobic core including several key hydrophobic residues. A key design feature of BBA1 is the utilization of a type II‘ reverse turn to promote β-hairpin formation; a control peptide, in which the β-turn of BBA1 was changed from a type II‘ to a type II, lacks tertiary structure. Thus the effects of the turn type on the three-dimensional structure of this motif are dramatic. BBA1, a 23-residue mixed α/β motif, defines a new lower limit for the size of an independently folded polypeptide with native structure.