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Membrane Surface-Associated Helices Promote Lipid Interactions and Cellular Uptake of Human Calcitonin-Derived Cell Penetrating Peptides

Michael E. Herbig ^*, Kathrin Weller ^*, Ulrike Krauss , Annette G. Beck-Sickinger , Hans P. Merkle ^* and Oliver Zerbe 

^* Drug Formulation and Delivery Group, Department of Chemistry and Applied BioSciences, Swiss Federal Institute of Technology Zurich (ETH Zurich), CH-8093 Zurich, Switzerland; Institute of Biochemistry, University of Leipzig, D-04103 Leipzig, Germany; and Institute of Organic Chemistry, University of Zurich, CH-8057 Zürich, Switzerland

Correspondence: Address reprint requests to Oliver Zerbe, Institute of Organic Chemistry, University of Zurich, Ch-8057 Zürich, Switzerland. Tel.: 41-44-63542 63; Fax: 41-44-6356833; E-mail address: oliver.zerbe{at}oci.unizh.ch.

hCT(9-32) is a human calcitonin (hCT)-derived cell-penetrating peptide that has been shown to translocate the plasma membrane of mammalian cells. It has been suggested as a cellular carrier for drugs, green fluorescent protein, and plasmid DNA. Because of its temperature-dependent cellular translocation resulting in punctuated cytoplasmatic distribution, its uptake is likely to follow an endocytic pathway. To gain insight into the molecular orientation of hCT(9-32) when interacting with lipid models, and to learn more about its mode of action, various biophysical techniques from liposome partitioning to high-resolution NMR spectroscopy were utilized. Moreover, to establish the role of individual residues for the topology of its association with the lipid membrane, two mutants of hCT(9-32), i.e., W30-hCT(9-32) and A23-hCT(9-32), were also investigated. Although unstructured in aqueous solution, hCT(9-32) adopted two short helical stretches when bound to dodecylphosphocholine micelles, extending from Thr¹⁰ to Asn¹⁷ and from Gln²⁴ to Val²⁹. A23-hCT(9-32), in which the helix-breaking Pro²³ was replaced by Ala, displayed a continuous -helix extending from residue 12 to 26. Probing with the spin label 5-doxylstearate revealed that association with dodecylphosphocholine micelles was such that the helix engaged in parallel orientation to the micelle surface. Moreover, the Gly to Trp exchange in W30-hCT(9-32) resulted in a more stable anchoring of the C-terminal segment close to the interface, as reflected by a twofold increase in the partition coefficient in liposomes. Interestingly, tighter binding to model membranes was associated with an increase in the in vitro uptake in human cervix epithelial andenocarcinoma cell line cells. Liposome leakage studies excluded pore formation, and the punctuated fluorescence pattern of internalized peptide indicated vesicular localization and, in conclusion, strongly suggested an endocytic pathway of translocation.

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