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. 2020 Jan;112(1):e24125.
doi: 10.1002/pep2.24125. Epub 2019 Jun 12.

The effect of turn residues on the folding and cell-penetrating activity of β-hairpin peptides and applications toward protein delivery

Stephen E Miller  1 , Joel P Schneider  1
Affiliations

The effect of turn residues on the folding and cell-penetrating activity of β-hairpin peptides and applications toward protein delivery

Stephen E Miller et al. Pept Sci (Hoboken). 2020 Jan.

Abstract

Cell-penetrating peptides (CPPs) are useful tools for the delivery of a wide variety of cargo into cells. Our lab has developed two classes of CPPs based on β-hairpin sequences, one that folds at the surface of cell membranes and the other that is intrinsically disordered. Although these peptides can effectively deliver different types of cargo, their use in protein delivery has been hindered due to the presence of non-natural D-proline within the central turn region of both sequences, which prohibits functionalizing proteins with the CPPs via standard expression protocols. In this work, we describe new CPPs that replace the non-natural turn region with natural turn motifs amenable to protein expression. We first investigate how these changes within the turn affect various CPP-related properties in the absence of protein cargo, and then generate protein fusions for intracellular delivery.

Keywords: cell-penetrating peptides; protein delivery; β-hairpin.

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Figures

FIGURE 1
FIGURE 1
Expression-capable analogs of SVS1 (XS) and CLIP6 (XC). A, SVS1 folds at cell surfaces and enters through a mixture of uptake mechanisms, but can be lytic. CLIP6 is intrinsically disordered, is less toxic, and delivers small molecules through direct translocation. Analogs of SVS1 and CLIP6, utilizing natural turn residues, have altered CPP properties and can be fused to protein cargo through expression. B, Sequences for SVS1, CLIP6, and natural turn containing analogs
FIGURE 2
FIGURE 2
Turn residues affect folding and activity. A, CD spectra in the presence of negatively charged POPC/POPS liposomes. B, Cytotoxicity toward A549 cells. C, Uptake of fluorescein-tagged peptides in A549, with and without the presence of cell-uptake inhibitors. ****P < .0001; ***P < .001; **P < .01; ns = not significant. For interpeptide P values, see Table S1
FIGURE 3
FIGURE 3
Generation of EGFP-CPP analogs. A, Proteins are expressed from E. coli with an N-terminal His-tag, that is cleaved following immobilized metal ion affinity chromatography (IMAC). B, SDS-PAGE gels of purified proteins after His-tag removal
FIGURE 4
FIGURE 4
Assessment of protein delivery into A549 cells by (A) flow cytometry and (B) confocal microscopy. ****P < .0001; ***P < .001; **P < .01; ns = not significant
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