An engineered tryptophan zipper‐type peptide as a molecular recognition scaffold

Abstract

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.

Aromatic interactions with naphthylalanine in a β‐hairpin peptide

“…The stability gained from tryptophan pairs has led to their wide application [6][7][8][9]. To expand this motif, various unnatural residues have been used [10][11][12][13], which have conferred advantages, such as reduced proteolysis, over tryptophan [14].…”

Using Thioamides To Site-Specifically Interrogate the Dynamics of Hydrogen Bond Formation in β-Sheet Folding

“…While there are a large number of experimental studies on the folding mechanism of β-hairpins, − a direct assessment of interstrand H-bond formation in the folding transition state of β-hairpins, to the best of our knowledge, has never been done before. Specifically, we chose to study a variant of well-studied β-hairpins, tryptophan zippers (Trpzips), due to the large body of experimental and computational research − on their folding thermodynamics, kinetics, and mechanisms. As shown (Figure ), this Trpzip variant (Trpzip-2c following Keiderling and co-workers’ sequence: NH 2 -AWAWENGKWAWA-CONH 2 ) folds into an antiparallel β-sheet structure that is stabilized by several interactions, including six BB-HBs, among which three are perturbed in the current study by individually substituting Ala1, Ala10, and Glu5 with their thioamide derivatives, i.e., thioalanine (TA) and thioglutamate (TE), and the corresponding mutants are hereafter referred to as A1/TA, A10/TA, and E5/TE, respectively.…”

Small and Simple, yet Sturdy: Conformationally Constrained Peptides with Remarkable Properties

“…Cystine-based macrocycles are, however, prone to reductive opening and associated with rather high flexibility due to significant rotational freedom of disulfide bond. Another approach relies on edge-to-face π-π stacking interactions between the cross-strand tryptophan residues located at non-hydrogen-bonding positions (called the tryptophan zipper motifs) to stabilize the antiparallel β-strand pairing [74,75]. Both rigidification strategies are compatible with biological peptide display libraries as they depend on proteinogenic amino acid residues as stabilizers.…”