DESIGN AND NMR ANALYSES OF COMPACT, INDEPENDENTLY FOLDED BBA MOTIFS

Background: Small folded polypeptide motifs represent highly simplifie d systems for theoretical and experimental studies on protein structur e and folding. We have recently reported the design and characterizati on of a metal-ion-independent 23-residue peptide with a beta beta alph a structure (BBA1), based on the zinc finger domains. To understand be tter the determinants of structure for this small peptide, we investig ated the conformational role of the synthetic residue 3-(1,10-phenanth rol-2-yl)-L-alanine (Fen) in BBA1. Results: NMR analysis revealed that replacing the Fen residue of peptide BBA1 by either of the natural am ino acids tyrosine (BBA2) or tryptophan (BBA3) resulted in conformatio nal flexibility in the sheet and loop regions of the structure. This c onformational ambiguity was eliminated in peptides BBA4 and BBA5 by in cluding charged residues on the exterior of the beta hairpin designed to both select against the undesired fold and stabilize the desired st ructure. The evaluation of two additional peptides (BBA6 and BBA7) pro vided further insight into the specific involvement of the surface pol ar residues in the creation of well-defined structure in BBA4 and BBA5 , The sequences of BBA5, BBA6 and BBA7 include only one non-standard a mino acid (D-proline), which constrains a critical engineered type II' turn. Conclusions: Manipulation of residues on the exterior of small beta beta alpha motifs has led to the design of 23-residue polypeptide s that adopt a defined tertiary structure in the absence of synthetic amino acids, increasing the availability and expanding the potential u ses of the BBA motif, The importance of negative design concepts to th e creation of structured polypeptides is also highlighted.