(272f) Rapid Uptake of Fluorescent Peptides into Intact Mammalian Cells Using a b-Hairpin Sequence Motif

Melvin, A., Louisiana State University
Safabakhsh, N., Louisiana State University
Pappas, G., Louisiana State University

Fluorescent, peptide-based reporters are an attractive method to quantify intracellular enzyme activity in intact cells due to their relative ease of synthesis and the fact that they do not require complex genetic engineering. While unstructured peptides are often rapidly degraded in the cytosol, structured sequences, such as a β-hairpin ‘protectides’, resist intracellular degradation which makes them ideal candidates for enzyme reporters. Recent work has verified the utility of incorporating a protectide on the N-terminus of a short peptide substrate to measure enzyme activity.1 One challenge in the use of peptide-based reporters is the efficient delivery of the peptide into intact cells. While this can be accomplished by external manipulation of the cells, a preferred method is to utilize a cell penetrating peptide (CPP) sequence to facilitate transport across the plasma membrane. This presentation highlights the identification and characterization of a short peptide sequence that incorporates a β-turn motif to act, not only as a protectide, but also as a CPP.  The protectide, named OWRWR [Ac-OWVRVpGO(FAM)WIRQ-NH­2], was demonstrated to rapidly permeate HeLa cells using both fluorometry and microscopy techniques at temperatures ranging from 4°C-37°C. The stability of OWRWR under conditions similar to the cytosolic environment was verified using a degradation assay and reverse phase high performance liquid chromatography (RP-HPLC). The β-turn sequence was confirmed using circular dichroism (CD). The internalization efficiency of OWRWR was compared to a commercially available CPPs and non-permeating control peptides. Interestingly, it was discovered that the identity of the N-terminal amino acid residue played an essential role in the ability of this protectide to function as a CPP. An ornithine (O) to arginine (R) substitution at the N-terminus completely eliminated the permeability of peptide into intact cells. As such, subsequent characterization of a library of protectide variants was performed to identify the impact of the identity of the positively charged residues in the β-turn sequence. Finally, the protectide was incorporated into the N-terminus of an enzyme substrate to generate a long-lived, cell permeable fluorescent probe for detecting intracellular enzyme activity.

 1.         Yang, S.; Proctor, A.; Cline, L.; Houston, K.; Waters, M.; Allbritton, N., beta-Turn sequences promote stability of peptide substrates for kinases within the cytosolic environment. Analyst 2013, 138 (15), 4305-4311.