An iterative design process involving the synthesis andstructural analyses of five polypeptides patternedafter the zinc finger domains is described. This process has ledto the development of a metal-independent folded
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motif,
BBA1. In contrast to the zinc fingersand other naturally occurring peptides of similar size, thissmallmonomeric structure folds without the assistance of metal cationligation or disulfide bridges. To probe the effectof metal binding on the secondary and tertiary structure of peptidesthroughout the design process, a non-standardamino acid 3-(1,10-phenanthrol-2-yl)-
L-alanine (Fen)was incorporated and its unique chromophore utilized forcirculardichroism (CD) analysis. Advanced designs were analyzed by both CDand 2D NMR. The solution structure of
BBA1 was determined using NOE restrained simulatedannealing. The average RMSD for the backbone atoms ofresidues 1-22 is 0.9 ± 0.3 Å. Analysis of the resultingstructure reveals that the
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-helix and
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-hairpin areassociated
via a well-defined hydrophobic core including several keyhydrophobic residues. A key design feature of
BBA1isthe utilization of a type II' reverse turn to promote
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-hairpinformation; a control peptide, in which the
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-turn of
BBA1 was changed from a type II' to a type II, lackstertiary structure. Thus the effects of the turn type onthethree-dimensional structure of this motif are dramatic.
BBA1, a 23-residue mixed
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/
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motif, defines a newlowerlimit for the size of an independently folded polypeptide with nativestructure.