An engineered tryptophan zipper-type peptide as a molecular recognition scaffold.
In an effort to develop a structured peptide scaffold that lacks a disulfide bond and is thus suitable for molecular recognition applications in the reducing environment of the cytosol, we investigated engineered versions of the trpzip class of beta-hairpin peptides. We have previously shown that even most highly folded members of the trpzip class (i.e. the 16mer peptide HP5W4) are substantially destabilized by the introduction of mutations in the turn region and therefore not an ideal peptide scaffold. To address this issue, we used a FRET-based live cell screening system to identify extended trpzip-type peptides with additional stabilizing interactions. One of the most promising of these extended trpzip-type variants is the 24mer xxtz1-peptide with the sequence KAWTHDWTWNPATGKWTWLWRKNK. A phage display library of this peptide with randomization of six residues with side chains directed towards one face of the hairpin was constructed and panned against immobilized streptavidin. We have also explored the use of xxtz1-peptide for the presentation of an unstructured peptide 'loop' inserted into the turn region. Although NMR analysis provided no direct evidence for structure in the xxtz1-peptide with the loop insertion, we did attempt to use this construct as a scaffold for phage display of randomized peptide libraries. Panning of the resulting libraries against streptavidin resulted in the identification of peptide sequences with submicromolar affinities. Interestingly, substitution of key residues in the hairpin-derived portion of the peptide resulted in a 400-fold decrease in K(d), suggesting that the hairpin-derived portion plays an important role in preorganization of the loop region for molecular recognition.
Cheng
Zihao
Z
Department of Chemistry, University of Alberta, Edmonton, Alberta T6G2G2, Canada.
Campbell
Robert E
RE
D031541:Fluorescence Resonance Energy Transfer; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*
D019151:Peptide Library; D010455:Peptides; D014364:Tryptophan