In Vivo Screening Identifies a Highly Folded β‐Hairpin Peptide with a Structured Extension

Abstract

The conventional approach for developing folded peptides involves chemical synthesis of systematically modified peptide variants and individual characterization by circular dichroism and NMR spectroscopy. [1][2][3][4][5] The use of synthetic peptides and reliance on low-throughput characterization techniques necessarily restricts the sequence diversity that can be explored, though efforts have been made to overcome this limitation. [6][7][8] We have recently described an approach that enables us to asses the ability of peptides to fold into b-hairpins in the cytoplasm of live cells.[9] Our strategy entails the recombinant expression of a peptide gene fused in frame between flanking genes encoding a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP). If a particular peptide sequence adopts a folded structure, CFP and YFP are brought into closer proximity and exhibit a higher efficiency of fluorescence resonance energy transfer (FRET). Higher FRET efficiency enhances the YFP (acceptor) fluorescence at the expense of the CFP (donor) fluorescence. Imaging of plates harboring colonies of transformed bacteria provides the YFP and CFP fluorescence emission intensities for each colony, and thus peptides that are highly folded in vivo can be distinguished from those that are not.As will be described in this manuscript, this FRET-based approach also provides a versatile method for screening large libraries of peptide sequences for highly structured variants. We have applied this strategy to the development of a version of a "tryptophan zipper" (trpzip)-type b-hairpin [1] with a structured extension. Trpzips are a class of highly folded b-hairpins that are defined by the presence of cross-strand diagonally oriented Trp/Trp pairs on one face of the hairpin. [1,5,10] The minimal and highly stable trpzip structure has emerged as a preferred model system for computational and experimental studies of protein folding. [11][12][13] We have proposed that trpzip-type peptides (or tandem fusions of such peptides) could serve as a minimal protein scaffold for molecular recognition in the cytoplasm of live cells. [9, 14] With this goal in mind, we sought to employ our screening strategy to identify a candidate trpziptype peptide with high fold stability in vivo. A similar approach has previously been used to increase the thermal stability of engineered immunoglobulin V L domains. [15] The template for our initial "extended trpzip" library was a 20-mer version of the highly folded 16-mer trpzip HP5W4.[10]The 20-mer contained two additional pairs of residues, one random and one threonine, genetically inserted after what would otherwise have been the second and 14th residues of HP5W4. ; here X represents all 20 amino acids, and the residues inserted relative to HP5W4 are underlined. Assuming b-strand conformation, the randomized positions would be directed towards the face of HP5W4 that harbors the interdigitated Trp side chains.Escherichia coli was transformed with the gene library, and 6 10 3 colonies on 10 Petri dis...

Stabilizing capping motif for β-hairpins and sheets

“…An unnatural π-cation interaction has also been shown to stabilize the turn in a tripeptide (24). There has been limited evidence for hairpin fold stabilization by hydrophobic effects involving terminal residues of hairpins (25)(26)(27): the stabilizing effects of cross-strand hydrophobic pairs has been reported to decrease with increasing contact order (28).…”

A Structuring Repeat for Peptide Design: Long Beta Ribbons

“…Cross‐strand Trp/Trp pairs do not associate to form sheet‐stabilizing edge‐to‐face indole–indole interactions at interior strand positions. This intolerance for mid‐strand positions has been demonstrated repeatedly; for example, by swapping a Trp/Trp cap to an interior position, or by extensions of the β‐sheet of trpzip which fail to adopt β‐sheet conformations …”

Separation of peptide fragments of a protein kinase C substrate fused to a β-hairpin by capillary electrophoresis

“…β-Hairpin peptides comprising only α-amino acids have also demonstrated increased resistance to proteases in vitro [14]. More recently, well-folded β-hairpins that exhibit increased resistance to protease and peptidase degradation have been developed [16–18]. The presence of the turn-inducing sequence, typically Asn-Gly or dPro-Gly, and the cross-strand interactions, especially aromatic and hydrophobic interactions, play an important role in stability of the β-hairpin and consequently in its resistance to peptidases [14, 17, 18].…”

“…More recently, well-folded β-hairpins that exhibit increased resistance to protease and peptidase degradation have been developed [16–18]. The presence of the turn-inducing sequence, typically Asn-Gly or dPro-Gly, and the cross-strand interactions, especially aromatic and hydrophobic interactions, play an important role in stability of the β-hairpin and consequently in its resistance to peptidases [14, 17, 18]. A peptide having the amino acid sequence Ac-RWVKVNGOWIKQ-NH 2 (here-in referred to as “WKWK”) has proven to be highly resistant to degradation by trypsin, α-chymotrypsin and Pronase E compared to a scrambled peptide control [14].…”